Heat stable colloidal iron oxides coated with reduced carbohydrates and uses thereof

ABSTRACT

Compositions, methods of making the compositions, and methods of using the compositions are provided for an enhanced magnetic resonance imaging agent and a hematinic agent, the agents comprising carboxyalkylated reduced polysaccharides coated ultrasmall superparamagnetic iron oxides. Methods of use of the carboxymethyl reduced dextran as a plasma extender are provided.

RELATED APPLICATIONS

[0001] This application is a divisional application from U.S. patentapplication Ser. No. 09/521,264, filed Mar. 8, 2000 which in turn claimsthe benefit of Provisional Application No. 60/128,579, filed in theUnited States Patent and Trademark Office on Apr. 9, 1999, both of whichare hereby incorporated by reference herein.

TECHNICAL FIELD

[0002] The field relates to compositions which are carboxymethyl reducedpolysaccharides, and methods for use as plasma extenders and for coatingiron oxide particles, and compositions comprised of superparamagneticand non-superparamagnetic iron oxides coated with a reducedpolysaccharide or derivatized reduced polysaccharide, and methods foruse as MRI contrast agents and hematinics.

BACKGROUND

[0003] Since the invention of magnetic resonance imaging (MRI), aparallel technology of injectable chemicals called contrast agents hasdeveloped. Contrast agents play an important role in the practice ofmedicine in that they help produce more useful MRI images for diagnosticpurposes. In particular, two classes of imaging agents have beendeveloped and adopted in clinical practice. These are: low molecularweight gadolinium complexes such as Magnavist®; and colloidal ironoxides such as Feridex I.V.® and Combidex®. Neither of these two typesof agents is ideal. Problems encountered with these agents are shown inTable 1, and include: expense of components; inefficiency of synthesis;loss of coating during terminal sterilization (autoclaving); narrowrange of organ uptake for purposes of imaging; toxic side-effects;restriction of use to either first pass or equilibrium dosing, andothers that are described herein. Agents that overcome these problems,and that combine the properties of these two types of contrast agents,are highly desirable. TABLE 1 Comparison of ideal properties of MRIcontrast agents with properties of low molecular weight gadolinium basedcontrast agents and colloidal iron oxides. Properties of an ideal lowmolecular weight colloidal iron contrast agent gadolinium oxides Lowproduction costs: Yes No efficient synthesis Autoclavable without Yes Noexcipients T1 agent Yes Sometimes T2 agent No Yes Non toxic Yes NoImaging vascular No No compartment at early phase (as a bolusadministration) and at a late stage (equilibrium phase) Multipleadministration in No No single examination Image of multiple target YesSometimes organs Bolus injection Yes No Low volume of injection No NoIron source for anemia No Yes

SUMMARY

[0004] An embodiment of the invention is a method of providing an ironoxide complex for administration to a mammal subject, the methodcomprising: producing a reduced polysaccharide iron oxide complex, andsterilizing the complex by autoclaving. In general, the reducedpolysaccharide is a reduced polymer of glucose. An example of a reducedpolymer of glucose is a reduced dextran. The reduced polysaccharide isproduced through reaction of a polysaccharide with a reagent selectedfrom the group consisting of a borohydride salt or hydrogen in thepresence of a hydrogenation catalyst. In a further aspect of the method,the iron oxide is superparamagnetic.

[0005] Another preferred embodiment of the invention is a method ofproviding an iron oxide complex for administration to a mammaliansubject, the method comprising: producing a derivatized reducedpolysaccharide iron oxide complex, and sterilizing the complex byautoclaving. According to this method, producing the complex can includederivatizing a reduced polysaccharide by carboxyalkylation, for example,wherein the carboxyalkylation is a carboxymethylation. Further accordingto this method, the reduced polysaccharide can be a reduced dextran. Thederivatized, reduced polysaccharide can be isolated as the sodium saltand does not contain an infrared absorption peak in the region of1650-1800 cm⁻¹. In one aspect of the method, producing the derivatizedreduced polysaccharide is achieved at a temperature of less thanapproximately 50° C. In another aspect of the method, producing thederivatized reduced polysaccharide is achieved at a temperature of lessthan approximately 40° C. In a further aspect of the method, the ironoxide is superparamagnetic.

[0006] In yet another embodiment, the invention provides a method offormulating an iron oxide complex coated with a reduced polysaccharide.This composition is for pharmacological use and the composition hasdecreased toxicity in comparison to an analogous iron oxide complexcoated with native polysaccharide. The method of formulating such aniron oxide complex comprises: producing a reduced polysaccharide ironoxide complex, and sterilizing the complex by autoclaving. Theformulation provides a polysaccharide which was produced by reacting thepolysaccharide with one of a reducing agent selected from the groupconsisting of a borohydride salt or hydrogen in the presence of anhydrogenation catalyst. The reduced polysaccharide iron oxide complexhaving such decreased toxicity. In a further aspect of the method, theiron oxide is superparamagnetic.

[0007] In yet another embodiment, the invention provides a method offormulating an iron oxide complex coated with a reduced derivatizedpolysaccharide. This composition is for pharmacological use and thecomposition has decreased toxicity in comparison to an analogous ironoxide complex coated with native derivatized polysaccharide. The methodof formulating such an iron oxide complex comprises: producing a reducedderivatized polysaccharide iron oxide complex; and sterilizing thecomplex by autoclaving. According to this method, producing the complexcan include derivatizing a reduced polysaccharide by carboxyalkylation,for example, wherein the carboxyalkylation is a carboxymethylation.Further according to this method, the reduced polysaccharide can be areduced dextran. The derivatized, reduced polysaccharide can be isolatedas the sodium salt and does not contain an infrared absorption peak inthe region of 1650-1800 cm⁻¹. In one aspect of the method, producing thederivatized reduced polysaccharide is achieved at a temperature of lessthan approximately 50° C. In another aspect of the method, producing thederivatized reduced polysaccharide is achieved at a temperature of lessthan approximately 40° C. In a further aspect of the method, the ironoxide is superparamagnetic.

[0008] Another embodiment of the invention provides a reducedderivatized polysaccharide iron oxide complex with T1 and T2 relaxationproperties to allow contrast agent signal enhancement with T1 sequencesand signal diminishment with T2 sequences. A further aspect of theembodiment is that the reduced derivatized polysaccharide iron oxide canbe administered multiple times for sequential imaging in a singleexamination. Yet another aspect of the agent is that it can be used toimage multiple organ systems including the vascular system, liver,spleen, bone marrow, and lymph nodes.

[0009] Another embodiment of the invention provides a reducedpolysaccharide iron oxide complex for use as an intravenous ironsupplement.

[0010] Another embodiment of the invention provides a reducedderivatized polysaccharide iron oxide complex for use as an intravenousiron supplement.

[0011] In yet a further embodiment, the invention provides an improvedmethod of administering to a mammalian subject an autoclaved reducedpolysaccharide iron oxide complex. The improved method of administrationcomprising: injection of an autoclaved reduced polysaccharide iron oxidecomplex in a volume of 15 mL or less. In another aspect of theembodiment the injected volume is injected as a bolus. In a furtheraspect of the method, the iron oxide is superparamagnetic. In a furtheraspect of the embodiment the injected volume provides improved imagequality.

[0012] In yet a further embodiment, the invention provides an improvedmethod of administering to a mammalian subject an autoclaved derivatizedreduced polysaccharide iron oxide complex. The improved method ofadministration comprising: injection of an autoclaved reducedderivatized polysaccharide iron oxide complex in a volume of 15 mL orless. In another aspect of the embodiment the injected volume isinjected as a bolus. In a further aspect of the method, the iron oxideis superparamagnetic. In a further aspect of the embodiment the injectedvolume provides improved image quality.

[0013] An embodiment of the invention provides an improved method ofadministering to a mammalian subject a reduced polysaccharide ironcomplex in a manner that the composition provides reduced toxicity,wherein the improvement comprises utilizing a reduced polysaccharide informulation of the composition. In a further aspect of the embodiment,the iron oxide is superparamagnetic.

[0014] An embodiment of the invention provides an improved method ofadministering to a mammalian subject a reduced derivatizedpolysaccharide iron complex in a manner that the composition providesreduced toxicity, wherein the improvement comprises utilizing a reducedderivatized polysaccharide in formulation of the composition. In afurther aspect of the embodiment, the iron oxide is superparamagnetic.

[0015] An embodiment of the invention provides a reduced polysaccharideiron oxide complex, wherein the reduced polysaccharide is derivatized,for example, the reduced derivatized polysaccharide is a carboxyalkylpolysaccharide. The carboxyalkyl is selected from the group consistingof carboxymethyl, carboxyethyl and carboxypropyl. Further, the reducedpolysaccharide can be a reduced dextran, for example, the reduceddextran can be a reduced carboxymethyl dextran. A further aspect of thisembodiment of the invention is that the level of derivatization of thereduced dextran is at least 750 μmole but less than 1500 μmole ofcarboxyl groups per gram of polysaccharide wherein said composition hasreduced toxicity relative to composition with respect to lower levels ofderivatization.

[0016] An embodiment of the invention provides a reduced polysaccharideiron oxide complex, such complex being stable at a temperature of atleast approximately 100° C. In a preferred embodiment, such complex isstable at a temperature of approximately 121° C. In an even morepreferred aspect of the reduced polysaccharide iron oxide complex, suchcomplex is stable at a temperature of at least 121° C. for a timesufficient to sterilize the complex. In a further aspect of theembodiment, the iron oxide is superparamagnetic.

[0017] An embodiment of the invention provides a reduced derivatizedpolysaccharide iron oxide complex, such complex being stable at atemperature of at least approximately 100° C. In a preferred embodiment,such complex is stable at a temperature of approximately 121° C. In aneven more preferred aspect of the reduced polysaccharide iron oxidecomplex, such complex is stable at a temperature of at least 121° C. fora time sufficient to sterilize the complex. In a further aspect of theembodiment, the iron oxide is superparamagnetic.

[0018] A preferred embodiment of the invention is a method offormulating for pharmacological use a reduced polysaccharide iron oxidecomplex having increased pH stability in comparison to the correspondingnative dextran iron oxide, the method comprising: providing dextran; andreacting the dextran with a borohydride salt or hydrogen in the presenceof an hydrogenation catalyst, reacting the reduced dextran with ironsalts to provide a formulation having a stable pH.

[0019] A preferred embodiment of the invention is a method offormulating for pharmacological use a reduced derivatized polysaccharideiron oxide complex having increased pH stability in comparison to thecorresponding native dextran iron oxide, the method comprising:providing dextran; and reacting the dextran with a borohydride salt orhydrogen in the presence of an hydrogenation catalyst, reacting thereduced dextran with iron salts to provide a formulation having a stablepH.

[0020] In another embodiment, the invention provides a method offormulating a reduced derivatized dextran composition forpharmacological use wherein the composition has decreased toxicity incomparison to native dextran, comprising: producing a reducedderivatized polysaccharide; and sterilizing the product by autoclaving.According to this method, the reduced polysaccharide is obtained byreacting the native polysaccharide with one of several reducing agentsselected from the group consisting of a borohydride salt, or hydrogen inthe presence of a hydrogenation catalyst. In a preferred aspect of theembodiment the polysaccharide is dextran. Producing the composition caninclude derivatizing a reduced polysaccharide by carboxyalkylation, forexample, wherein the carboxyalkylation is a carboxymethylation. Furtheraccording to this method, the reduced polysaccharide can be a reduceddextran. The derivatized, reduced polysaccharide can be isolated as thesodium salt and does not contain an infrared absorption peak in theregion of 1650-1800 cm⁻¹. In one aspect of the method, producing thederivatized reduced polysaccharide is achieved at a temperature of lessthan approximately 50° C. In another aspect of the method, producing thederivatized reduced polysaccharide is achieved at a temperature of lessthan approximately 40° C.

[0021] An embodiment of the invention provides an improved method ofadministering to a mammalian subject a reduced derivatizedpolysaccharide in a manner that the composition provides reducedtoxicity, wherein the improvement comprises utilizing a reducedpolysaccharide in formulation of the composition.

[0022] An embodiment of the invention provides a reduced polysaccharide,wherein the reduced polysaccharide is derivatized, for example, thereduced derivatized polysaccharide is a carboxyalkyl polysaccharide. Thecarboxyalkyl is selected from the group consisting of carboxymethyl,carboxyethyl and carboxypropyl. Further, the reduced polysaccharide canbe a reduced dextran. A further aspect of this embodiment of theinvention is that the level of derivatization of the reduced dextran isat least 750 micromolar of carboxyl groups per gram of polysaccharidewherein said composition has reduced toxicity relative to compositionwith lower levels of derivatization.

[0023] Another embodiment of the invention is a method of formulating adextran composition for pharmacological use and having decreasedtoxicity in comparison to native dextran, the method comprising:providing dextran; and reacting the provided dextran with a borohydridesalt or hydrogen in the presence of an hydrogenation catalyst followedby carboxymethylation, the reduced carboxymethylated dextran havingdecreased toxicity.

[0024] Another embodiment of the invention is an improved method ofadministering to a mammalian subject a polysaccharide composition of thetype wherein the composition includes dextran in a manner that thecomposition provides reduced toxicity, wherein the improvement comprisesutilizing reduced carboxymethylated dextran in lieu of dextran in theformulation. In another aspect, an embodiment of the invention is animproved method of administering to a mammalian subject a polysaccharidein a manner that the composition provides reduced toxicity, wherein theimprovement comprises utilizing a reduced carboxymethylatedpolysaccharide in formulation of the composition.

[0025] An embodiment of the invention provides a method of use ofreduced derivatized dextrans as blood expanders.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026]FIG. 1 shows a Fourier transform infrared (FTIR) spectrographicanalysis of carboxymethyl reduced dextran (CMRD) sodium salt obtainedwith Example 5.

[0027]FIG. 2 shows an FTIR spectrographic analysis of sodium salt CMRDcoated ultrasmall superparamagnetic iron oxide (USPIO; see U.S. Pat. No.5,055,288) obtained in Example 31.

[0028]FIG. 3 is a graph that shows the amount of carboxymethyl groups(micromoles) per gram of product, on the ordinate, as a function of theamount of bromoacetic acid mg/gram used in reactions with reduceddextran starting material, on the abscissa. The graph is plotted fromthe data of Table 2.

[0029]FIG. 4 shows pharmacokinetics of CMRD coated USPIO in the blood ofthree male rats following intravenous administration of 2.2 mg of ironper kg body weight. Samples (0.25 mL) of blood were collected at thetimes indicated on the abcissa, and relaxation times were measured on aBrucker Minispec spectrometer.

[0030]FIG. 5 shows the graph used to determine a half-life (67 minutes)of CMRD coated USPIO in rat blood. The data of FIG. 4 were used togenerate the graph in FIG. 5. The half-life range of 61 to 75 minuteswas within the 95% confidence level.

[0031]FIG. 6 shows MRIs of a rat, pre-administration (A) andpost-administration (B) of contrast agents, anterior portion at top.CMRD coated USPIO (5 mg of iron per kg body weight) was administeredinto the femoral vein prior to taking the post administration contrastimage. The figure illustrates enhanced visualization of the heart andsurrounding arteries and veins caused by administration of CMRD coatedUSPIO. Imaging was performed using a General Electric 2 Tesla magneticresonance imager.

[0032]FIG. 7 shows MRI images of a pig, pre-administration (A) andpost-administration (B) of contrast agent, anterior portion at top. CMRDcoated USPIO (Example 31; 4 mg of iron per kg body weight) wasadministered into the femoral vein prior to taking the postadministration contrast image. The figure illustrates enhancedvisualization of the heart and surrounding arteries and veins caused byadministration of CMRD coated USPIO. Imaging was performed using aSiemans 1.5T Magnatom Vision magnetic resonance imager.

[0033]FIG. 8 shows MRI images of the anterior portion of a normal humansubject, pre-administration (A) and post-administration (B) of contrastimaging agent. CMRD coated USPIO (4 mg of iron per kg body weight) wasadministered as a bolus into a vein in the arm prior to taking the postcontrast image. Imaging was performed 15 to 30 minutes afteradministration of contrast agent. The image illustrates enhancedvisualization of the heart and surrounding arteries and veins.

[0034]FIG. 9 shows the blood clearance kinetics in humans of imagingagent. CMRD coated USPIO (4 mg of iron per kg body weight), wasadministered as a bolus into a vein in the arm prior to taking bloodsamples. Samples were analyzed for 1/T2 relaxation to determine theblood concentration of the CMRD coated USPIO. The graph shows CMRDcoated USPIO concentration (ordinate) as a function of time (abscissa).

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

[0035] Table 1 summarizes the characteristics of two classes of MRIcontrast agents that have been previously described, and shows acomparison of their characteristics to those of an ideal contrast agent.Agents of the invention embody the ideal characteristics, as shownherein.

[0036] Surprisingly, the development and synthesis of preparations ofultrasmall superparamagnetic iron oxide (USPIOs) coated withpolysaccharide reduced dextrans and derivatives of reduced dextrans,such as the agents with the desirable properties as shown herein, arederived from a change in the chemical nature of one constituent, dextranT10. This change involved reduction of the terminal aldehyde group to analcohol of the polysaccharide used in its synthesis to an alcohol(Scheme 1). Scheme 1 illustrates the chemical change in a polysaccharidesuch as dextran upon treatment with sodium borohydride. The hemiacetalform of the polysaccharide (structure 1) is in equilibrium with thealdehyde form of the polysaccharide (structure 2). Structure 2represents less than 0.01% of the equilibrium mixture (Brucker, G.(1974) Organic Chemistry: Amino Acids, Peptides and Carbohydrates.,Tankonykiado Press, Budapest, p. 991). Treatment of structure 2 withsodium borohydride results in its irreversible conversion to the linearpolyol form of the polysaccharide (structure 3). The dynamic equilibriumbetween structures 1 and 2 allows complete conversion, when treated withsodium borohydride, to the linear polyol (structure 3).

[0037] Dextran coated superparamagnetic iron oxide particles haveparticular interest as magnetic resonance imaging (MRI) contrast agentsbecause of their ability to enhance images of the liver and lymph.Feridex I.V.® (Advanced Magnetics, Inc., Cambridge Mass.) is a dextrancoated superparamagnetic iron oxide MRI contrast agent, and approved foruse in humans. Combidex® (Advanced Magnetics, Inc.) is a dextran coatedultrasmall superparamagnetic iron oxide (USPIO) which has completedPhase III clinical trials for both liver imaging and Phase III trialsfor lymph imaging. Combidex® has a smaller mean diameter (20 ηm) thanFeridex I.V.® (60 nm), which gives it a different biodistribution inhumans. Combidex® is made by addition of base to a solution of dextran,ferric chloride and ferrous chloride. The synthetic process comprisescombining the ingredients, heating, and purifying by ultrafiltration.However, the yield of dextran added to the particles in the reaction isinefficient. Pharmaceutical grade dextran is the most expensivecomponent of the Combidex® synthesis. A more efficient use of dextran inthe synthesis of Combidex® is desirable to lower production costs.

[0038] Terminal sterilization (autoclaving) is a preferred method ofsterilizing drugs for injection. However, many superparamagnetic ironoxide colloids that are used as MRI contrast agents are synthesized withpolymer coatings and coverings that influence the biodistribution andelimination of these colloids. Upon exposure to the heat for theduration of the autoclaving process, the polymer coating can becomedissociated from the iron oxide cores. The functional consequences ofpolymer dissociation from the iron oxide are physical changes in thematerial, such as clumping, biodistribution changes (changes in plasmahalf life), and changes in toxicity profile (potential increases inadverse events). For example, a substantial decrease in the pH of thesolution can be detected following autoclaving of iron dextranparticles, and the pH continues to fall upon further storage.

[0039] Several solutions to the problem of imparting resistance to heatstress have been described. Palmacci et al., U.S. Pat. No. 5,262,176,hereby incorporated herein by reference, used crosslinked dextran tostabilize the covering on the iron oxide particles prior to autoclaving.The crosslinking process uses noxious agents such as epichlorohydrin andepibromohydrin, which must be removed from the colloid after thecrosslinking reaction.

[0040] Methods of preventing clumping of the colloid induced by heatstress that have no effect on coating dissociation have also beendescribed. These methods generally include the use of excipients duringthe autoclaving process. Groman et al., U.S. Pat. No. 4,827,945, andLewis et al., U.S. Pat. No. 5,055,288, both patents hereby incorporatedherein by reference, use citrate to prevent clumping of the particleswhen the coating dissociates. However, the use of citrate in highconcentrations in combination with heat can cause toxicity. Groman etal., U.S. Pat. No. 5,102,652, hereby incorporated herein by reference,uses low molecular weight carbohydrates such as mannitol to preventclumping during autoclaving. These excipients increase the cost andcomplexity of manufacturing the product, yet do not solve the problem ofdissociation of the polymer from the iron particle.

[0041] Josephson et al., U.S. Pat. No. 5,160,726, hereby incorporatedherein by reference, avoids heat stress on the coating by using filtersterilization rather than heat to sterilize the colloid. Filtersterilization is expensive since both the sterilization process andcontainer, closure must be performed in a germ free environment.Additionally, filter sterilizing has a higher rate of failure than theprocess of autoclaving, which reflects the inability to obtain anenvironment for the filtration step that is entirely germ free.

[0042] Maruno et al., U.S. Pat. No. 5,204,457, describes acarboxymethyl-dextran coated particle with improved stability up to 80°C. for an extended period but does not teach use of terminalsterilization by autoclaving. Hasegawa et al. (Japan J. Appl. Phys.,Part 1, 37(3A): 1029-1032, 1998) describes carboxymethyl dextran coatediron particles with thermal stability at 80° C., but does not teach useof a carboxymethyl reduced dextran coated particle, nor of terminalsterilization by autoclaving.

[0043] Magnetic resonance imaging agents act by affecting the normalrelaxation times, principally on the protons of water. There are twotypes of relaxation, one known as spin-spin or T1 relaxation, and thesecond known as spin-lattice or T2 relaxation. T1 relaxation generallyresults in a brightening of the image caused by an increase in signal.T1 processes are most useful in imaging of the vascular system. T2relaxation generally results in a darkening of the image caused by adecrease in signal. T2 processes are most useful in imaging of organssuch as the liver, spleen, or lymph nodes that contain lesions such astumors. All contrast agents have both T1 and T2 properties; however,either T1 or T2 relaxation can characterize the dominant relaxationproperty of a particular contrast agent. Low molecular weight gadoliniumbased contrast agents are T1 agents, and have primary application in theimaging of vascular related medical problems such as stroke andaneurysms and the brain. Iron oxide based colloidal contrast agents areT2 agents, and have primary application in imaging tumors of the liverand lymph nodes (prostate and breast cancer). An agent possessing bothT1 and T2 properties would be desirable. Using such an agent would (I)provide a single drug for all applications, and simplify the inventoryof the pharmacy, (ii) simplify imaging in the MRI suite, and (iii)improve patient care by permitting simultaneous examination of multiplemedical problems in a single patient during a single examination, ratherthan requiring use of either a T1 or a T2 contrast agent.

[0044] A dextran can elicit a sometimes fatal anaphylactic response whenadministered intravenously (i.v.) in man (Briseid, G. et al., ActaPharmcol. et Toxicol., 1980, 47:119-126; Hedin, H. et al., Int. Arch.Allergy and Immunol., 1997:113:358-359). Related adverse reactions havebeen observed also on administration of magnetic dextran coated ironoxide colloids. Non-magnetic dextran coated iron oxide colloids thathave utility as hematinics, particularly as an adjunct to erythropoietintreatment for end stage renal dialysis patients, can also have similarside effects.

[0045] Information regarding anatomical features within the vascularsystem can be obtained using contrast agents in two ways. When thecontrast agent is first administered as a bolus, it initially passesthrough the vascular tree as a relatively coherent mass. Coordinatingthe time of imaging of the desired anatomical feature to the time whenthe bolus passes through that feature can provide useful information.This technique of contrast agent use is called first pass imaging. At alater time, the bolus has been diluted by mixing, and attains anequilibrium concentration in the vascular system. Under certaincircumstances, this equilibrium or steady state can offer usefulinformation. Imaging can be performed at an early phase, within minutesafter injection of the contrast agent (“first pass”), and at a laterphase, from about ten minutes after injection of the contrast agent(equilibrium phase). Gadolinium agents are suited only for first passimaging due to their ready diffusion from the vascular system into theinterstitial spaces of the tissues. Previously described colloidal ironoxides are useful for the equilibrium due to their requirement fordilute administration over a prolonged time period. Colloidal ironoxides do not leak into the interstitial space but can remain in thevascular system for hours. An agent offering the opportunity to performboth first pass imaging and equilibrium imaging would be desirable.

[0046] During administration in a medical setting of a contrast agentfor “first pass” imaging, the timing of imaging and passage of the“first pass” of the contrast agent may not coincide. If a useful imagewas not obtained, it becomes desirable to administer a second dose ofcontrast agent to obtain another “first pass” image. On other occasionsradiologists find it useful to examine several volumes within thepatient requiring a multiple dosing regimen of contrast agent in orderto obtain “first pass” images at each of multiple sites of interest.With gadolinium contrast agents, this multiple administration “firstpass” application is not possible because the gadolinium leaks out ofthe vascular space producing a fuzzy background around blood vessels ofinterest. Current iron oxide colloidal based contrast agents are notsuitable as they are administered not as a bolus, but as a dilutesolution over a long time, obviating “first pass” applications.

[0047] Diagnosis of tumor progression in cancer patients is importantfor characterizing the stage of the disease, and for assessingtreatment. To minimize cost and discomfort to the patient, it isdesirable in an MRI examination to administer a single dose of contrastagent that would allow assessment of multiple organ systems that mightbe affected by the disease. For instance, in primary breast cancer, itis desirable to assess tumor status in the breast and at multiplemetastatic sites including the liver, spleen, bone marrow, and lymphnodes. Administration of gadolinium based contrast agents can notsatisfy this requirement due to their short half life in the body, theirleakage into the vascular system, and their inability to concentratewithin organs of interest. Iron oxide colloid based contrast agents suchas Combidex® can serve in this multiple capacity while Feridex I.V.®,another iron oxide colloid contrast agent, is limited to imaging theliver and the spleen.

[0048] Administration of a contrast agent in a small volume (less then 5ml) is desirable, as small volume administration improves the resolutionobtained from first pass imaging, and minimizes injection time anddiscomfort to the patient. Gadolinium based contrast agents areadministered in volumes of about 30 mL due to constraints caused by thesolubility and potency of these agents. Currently, iron oxide basedcontrast agents are administered as a dilute solution in a large volume(50-100 ml) over an extended period of time (30 minutes). Theseconstraints arise from safety issues associated with the rapid andconcentrated administration of iron oxide based agents. Bolus injectionis desirable in that it allows first pass imaging and shortens contacttime between the patient and health care provider. Further bolusinjection allows the practitioner to administer the contrast agent whilethe subject is in the MRI apparatus during the examination, therebyoptimizing efficient use of instrument imaging time. Gadolinium basedagents can be administered as a bolus.

[0049] Gadolinium based contrast agents consist of a chelating moleculeand the gadolinium cation. Gadolinium is a toxic element and must beexcreted from the body to avoid toxicity. Colloidal iron oxides are notexcreted from the body but are processed in the liver and other organsto metabolic iron, such as the iron in hemoglobin. Thus, compositions ofthe invention can serve as an iron supplement for patients sufferingfrom anemia, and are especially useful for patients undergoing treatmentwith erythropoietin.

[0050] An embodiment of the invention provides a method for thesynthesis of a colloid of an iron oxide associated with a water solublepolysaccharide coating in a manner that mitigates dissociation of thecoating from the iron oxide when the material is subjected to heatstress.

[0051] As used herein and in the accompanying claims, “heat stress” isdefined as heating the colloid to approximately 121° C. or higher forabout 30 minutes at neutral pH, or other combinations of time,temperature, and pH that are well known in the art to autoclave (orterminally sterilize) an injectable drug.

[0052] A method that is an embodiment of the invention includes thesteps of treating a polysaccharide with a reducing agent such aborohydride salt or with hydrogen in the presence of an appropriatehydrogenation catalyst such Pt or Pd to obtain the reducedpolysaccharide, such that the terminal reducing sugar has been reducedto give an open chain polyhydric structure. The reduced polysaccharidemay be an arabinogalactan, a starch, a cellulose, an hydroxyethyl starch(HES), an inulin or a dextran. Moreover, the polysaccharide may befurther functionalized prior to particle formation. The method furthercomprises mixing the reduced polysaccharide with iron salts in an acidicsolution selected from the group comprising ferric salts, ferrous salts,or a mixture of ferrous and ferric salts, cooling the solution,neutralizing the solution with a base, and recovering the coated ironoxide colloid.

[0053] In accordance with a further embodiment of the invention, thebases which may be employed are sodium hydroxide, sodium carbonate andmore preferably, ammonium hydroxide, for the step of neutralizing thecolloid. In a further embodiment of the invention, the polysaccharidederivative is reduced dextran and the iron salts may be ferrous andferric salts, which produce a superparamagnetic iron oxide colloid witha water soluble coating that does not dissociate from the iron oxidecore under heat stress during terminal sterilization.

[0054] In another embodiment of the invention, only ferric salts areemployed, yielding a non-superparamagnetic particle.

[0055] In another embodiment, a coated colloid may be prepared by addinga polysaccharide to an iron oxide sol (a colloidal dispersion in aliquid), adjusting the pH to 6-8 and recovering the coated iron oxidecolloid.

[0056] The term “colloid” as used in this specification and theaccompanying claims shall include any macromolecule or particle having asize less than about 250 nm. The iron oxide polysaccharide colloids ofthe invention have substantially improved physical characteristics andmanufacturability compared to previously described materials. Improvedphysical characteristics are evident in the ability of the colloid towithstand heat stress, as measured by subjecting the colloid to atemperature of 121° C. for 30 minutes. Colloid particles made accordingto the invention show less evidence of polysaccharide dissociation understress, remaining colloidal, and exhibiting no appreciable change insize. An example of a colloid with an unstable polysaccharide coatingincludes Combidex®, which when subjected to heat stress, lost 43% of itsdextran coating, and increased in particle diameter size from 21 ηm to587 nm; significant clumping of material was observed upon visualanalysis. Another superparamagnetic iron oxide dextran colloid,Feridex®, prepared according to U.S. Pat. No. 4,770,183, also exhibitedincreased particle size, as demonstrated by the inability of the heattreated colloid to pass through a filter having a 0.8 μm pore size,after a heat treatment comprising only 30 minutes at 121° C.

[0057] During manufacture, the process that is an embodiment of theinvention typically uses one tenth or less the amount of polysaccharidecompared to the amount required in previous preparations usingnon-reduced polysaccharide, resulting in substantial raw materials costsavings due to the improved efficiency of the process of the invention.

[0058] Variation in such factors as polysaccharide derivativeconcentration, base concentration and/or Fe(III)/Fe(II) concentrationcan produce colloids with different magnetic susceptibilities and sizes.Changing the Fe(III)/Fe(II) ratios changes the particle size and altersthe magnetic susceptibility. Higher ratios (for example, 2.0 mol/mol)tend to decrease susceptibility, whereas lower ratios (for example, lessthan 1.5 mol/mol) tend to increase particle size.

[0059] The process may be adjusted to yield colloids with differentbiological properties by changing the type of polysaccharide, andfurther derivatizing the particle after synthesis.

[0060] The colloids that are an embodiment of the invention can be usedas contrast agents for magnetic resonance imaging (MRI) or in otherapplications such as magnetic fractionation of cells, immunoassays,magnetically targeted drug delivery, and as therapeutic injectable ironsupplements. These colloids are particularly suited to parenteraladministration, because the final sterilization typically isautoclaving, a preferred method since it eliminates viability of allcellular life forms including bacterial spores, and viruses.

[0061] Previous methods for making colloids required the addition ofexcipients such as citrate or low molecular weight polysaccharides asstabilizers during the autoclaving process (see U.S. Pat. No. 4,827,945and U.S. Pat. No. 5,102,652), or avoided heat stress altogether by useof filter sterilization (see U.S. Pat. No. 5,150,726). Thus, theembodiments of the present invention that are the methods forsynthesizing the colloids, and the embodiments of the present inventioncomprising the colloid compositions, provide utilities as significantlyimproved MRI contrast agents, and hematinic agents that are ironsupplements. The improvements provided in these agents over prior artare found in the following facts demonstrated in the examples herein:that the agents which are embodiments of the present invention are heatsterilizable by autoclaving, and are thus optimized for long-termstorage at ambient temperatures; that these agents do not require theaddition of excipients for maintenance of stability during thesterilization or storage processes; that the agents are non-toxic tomammals including humans; that an effective dose of the agents used forimaging is a smaller amount of material than the agents described in theart; and that the pharmacokinetics following administration are suchthat iterated successive doses administered after a brief interval afteradministration of a first dose can be used to obtain additional imagesduring a single clinical visit and use of the imaging apparatus.

[0062] In the case of dextran and derivatives thereof, the formulationsprepared by this method are less immuno-responsive in mammals, as shownby data obtained using a rat model, and in clinical trials in humansubjects. The dextran- and dextran derivative-coated iron particlesenhanced imaging of the heart, lungs, kidneys, and other organs andsystems in three mammalian species: rat, pig, and human. The dextran-and dextran derivative-coated iron particles can be used also ashematinic agents, to provide iron in a more efficiently absorbed formatthan is true of oral iron supplements, to groups of patients who arechronically iron-deprived, such as dialysis patients, cancer patients,gastroenteritis patients, and recipients of erythropoietin. Thederivatized reduced dextrans can be used also as plasma extenders,which, unlike blood and blood fractions, do not have to be cross-matchedimmunologically, and unlike human serum albumin preparation, can besterilized in a manner that destroys viruses, including strains ofhepatitis, CMV, and HIV, spongiform encephalitis, and other infectiousagents. The plasma extenders of the invention do not have to berefrigerated or stored away from light and heat, and are thusadvantageous in emergency medical situations, such as treatment of shockdue to loss of blood such as trauma, even in tropical climates.

[0063] Examples 1, 2 and 3 show the methods for making reduced dextransof type T1, T5, and T10, respectively. Example 4 describes preparationof reduced pullulan.

[0064] Examples 5-9 describe the synthesis of carboxymethyl reduceddextran T10 with varying degrees of carboxymethylation, from nativedextran T10 (Table 2).

[0065] Examples 10-15 describe the synthesis of carboxymethyl reduceddextran T10with varying degrees of carboxymethylation, starting withreduced dextran T10 (Table 3).

[0066] Examples 16-18 describe the synthesis of carboxymethyl dextranT10, T40, and T70 from native dextran.

[0067] Examples 19-26 describe the preparation of reduced and nativedextran coated iron oxides. The conditions of the reactions in theseexamples were chosen to yield USPIOs coated either with reduced ornon-reduced polysaccharides. The reactions conditions for the nativedextran iron oxide preparations were the same as for the reduced dextranpreparations of the same molecular weights, to allow comparison of theeffectiveness of the respective dextrans in coating particles. Meanvolume diameter (MVD) and magnetic susceptibility of iron oxidepreparations obtained using reduced in comparison to nativepolysaccharides (prepared in these examples) are summarized in Table 4.

[0068] Examples 27-29 describe a procedure for the preparation of USPIOswith native T1, T5, and T10 dextrans, to obtain iron oxide colloidshaving a particle diameter of less than 30 nm. A comparison of effectsof native dextrans (Examples 27-29) and their respective reduceddextrans (Examples 19, 21, and 23) in the synthesis and properties ofiron oxide colloids is shown in Table 5.

[0069] Examples 30-31 describe the preparation USPIOs coated withcarboxymethyl native dextran T10 and carboxymethyl reduced dextran T10.

[0070] Examples 32-41 describe the preparation of USPIOs coated withcarboxymethyl reduced dextran T10 preparations containing varyingextents of carboxymethylation. The effect of extent ofcarboxymethylation of CMRDs on colloid size of USPIOs is shown in Table6. The effect of extent of carboxymethylation of CMRDs on solubility offerric/ferrous chloride solutions is shown in Table 7.

[0071] Examples 42-48 describe the synthesis of iron oxide sols andtheir stabilization with native and reduced dextrans and CMRD. Example49 describes preparation of CMRD coated non-magnetic iron oxide colloidusing base precipitation of ferric chloride and CMRD.

[0072] Example 50 examines the effect of the process of sterilization byautoclaving of various preparations of USPIOs coated with reduced andnative dextrans on the properties of these particles. The results areshown in Tables 8 and 9.

[0073] Example 51 reports the relaxation properties of various contrastagents comparing these properties for gadolinium based contrast agentsand USPIOs prepared with native dextran and carboxymethyl reduceddextran T10 (Table 10).

[0074] In Examples 52-53, the presence of symptoms of toxicity to ratsof reduced and non-reduced (native) dextran coated USPIOs wasdetermined, with response to an anaphylactic type reaction. The extentof the anaphylactic type reaction is determined by volume of paw edema.Similar studies were performed using native, reduced, andcarboxymethylated reduced dextrans. The results are summarized Tables11-14.

[0075] Example 54 and FIGS. 4 and 5 show the kinetics of clearance of aCMRD coated USPIO from rat circulation. The half-life of the agent isdetermined.

[0076] An enhanced MRI scan is shown in Example 55 and FIG. 6 followingadministration of CMRD coated USPIO, the scan showing images of the ratheart, aorta and other cardiac-associated arteries. Example 56 and FIG.7 show a CMRD coated USPIO enhanced MRI scan of the anterior portion ofa pig. Example 57 shows that injection of CMRD coated USPIOs into humansubjects, as part of a clinical trial, produced no adverse effects.Example 57 describes the biodistribution (FIG. 8), imaging kinetics(FIG. 9 and Table 15), and absence of background in MRI usage of thismaterial in humans. The data in this example show the ability of thepractitioner of the invention to perform multiple administrations andobtain subsequent images within the real time of an office visit orvisit to a MRI facility.

EXAMPLES General Procedures for the Synthesis of ReducedPolysaccharides.

[0077] Reduced polysaccharides were prepared by treatment with excesssodium borohydride and generally purified using five cycles ofultrafiltration. Distilled water is used throughout the examples. In thecase of the polysaccharide pullulan, the reduction mixture was usedwithout further purification. In all cases, the products showed lessthan 5% residual aldehyde content. Residual aldehyde concentration wasdetermined using a modified tetrazolium blue assay (Jue, C. K. et al.,J. Biochem. Biophys. Methods, 1985, 11:109-15). Dextran concentrationwas determined by a phenol/sulfuric acid assay (Kitchen,. R., Proc.Sugar Process. Res. Conf., 1983, 232-47). In cases where ultrafiltrationwas omitted, it was demonstrated that, except for the dextran T1, theresidual borate salts did not affect particle formation. Examples 1through 4 provide methods of synthesis of reduce polysaccharides T1, T5,and T10 dextrans, and pullulan, respectively. Retention times weredetermined using a Waters Ultrahydrogel 250 column, SN T52262A33, with20 mM phosphate buffered saline, 0.4 mL/min flow rate.

EXAMPLE 1

[0078] Reduced Dextran T1.

[0079] Dextran T1 (10 g) was dissolved in 100 mL water at 25° C., 1.0 gof sodium borohydride was added, and the mixture was stirred for 12 h.The pH was brought to 5.0 using 6 M HCl, and 200 mL ethanol (anhydrous)was added. The precipitate was collected by centrifugation. Theethanol/water layer was decanted, and the residue was dissolved in 100mL water. Addition of 200 mL of absolute ethanol was used to cause asecond precipitation, and the ethanol/water was again decanted. Theprecipitated product was dissolved in water, and was lyophilized toproduce a white solid, with a 60% yield. The observed HPLC retentiontimes (min) were: for reduced dextran, 24.4; and for native dextran,24.4.

Example 2

[0080] Reduced Dextran T5.

[0081] Dextran T5 (4 g) was dissolved in 25 mL water at 25° C., 83 mg ofsodium borohydride was added, and the mixture was stirred for 12 h. ThepH was brought to 5.0 using 6 M HCl. The mixture was ultrafilteredagainst a 1 kDa molecular weight cut-off (MWCO) membrane filter. Theproduct was lyophilized to produce a white solid, and a 70% yield wasobtained. The observed HPLC retention times (min) were: for reduceddextran, 22.9; for native dextran, 21.9.

Example 3

[0082] Reduced Dextran T10.

[0083] Dextran T10 (5,003 g) was dissolved in 26,011 g water. Sodiumborohydride was added (52.5 g) and the mixture was stirred for 24 hours.The pH was adjusted to 7.1 using 6 N HCl. The product was purified byrepeated ultrafiltration against a 3 kDa ultrafiltration membrane andlyophilized to produce a white solid. Yield: 3129 g. The observed HPLCretention times (min) were: for reduced dextran, 21.6; for nativedextran, 21.1.

Example 4

[0084] Reduced Pullulan.

[0085] Pullulan (90 mg) was dissolved in 0.8 mL water at 25° C., and 1mg of sodium borohydride was added. The mixture was stirred for 12 h,and was used directly in the preparation of USPIO.

[0086] General Procedures for Synthesis of a Carboxymethyl ReducedDextran Using Native Dextran T-10 as a Substrate.

[0087] Examples 5-9 describe the synthesis of carboxymethyl reduceddextrans from native dextran. Two general methods of synthesis arepresented, a low dextran concentration method (Example 5) in which thestarting concentration of native dextran was 70 mg/g, and a high dextranconcentration method (Examples 6-9), in which the starting concentrationof native dextran was 240 mg/g.

Example 5

[0088] Carboxymethyl Reduced Dextran T10 Prepared by the Low DextranConcentration Method.

[0089] The following solutions were prepared and cooled to 5° C.:Solution A contained 4,200 g sodium hydroxide in 10.5 liters of water;and Solution B contained 2,310 g bromoacetic acid in 5,700 mL water.Solution C contained 3,000 g dextran T10 in 7,500 mL water, heated to38° C.

[0090] Sodium hydroxide (600 g) was dissolved in 7.5 liters of water andwas warmed to 38° C. Sodium borohydride (60 g) was added and the mixturewas stirred for 2 min before adding Solution C, followed immediately byadding a second 60 g portion of sodium borohydride. The mixture wasstirred at 38° C. for 30 min, and then cooled to 15° C. Solution A wasadded, keeping the temperature of the solution below 25° C. Solution Bwas added, and the temperature of the solution was maintained below 25°C. The mixture was stirred for 2 hours at room temperature, and wasneutralized to pH 7.5 using 6M HCl cooled to 5° C., maintaining thesolution temperature below 35° C. The mixture was filtered though a 0.2μm filter, and diluted to 80 liters. The product was purified byrepeated ultrafiltration through a 3 kDa MWCO ultrafiltration membrane,again filtered through a 0.2 μm filter and was lyophilized.

[0091] The recovered solid, 2,560 g of carboxymethyl reduced dextran T10(sodium salt), showed a carboxyl content of approximately 1,265micromoles carboxyl per gram of product, as determined by titration. Theuse of bromoacetic acid allowed the reaction to proceed at a lowertemperature compared to use of chloroacetic acid, and produced a cleanerproduct as evidenced by its FTIR spectrum (FIG. 1). FIG. 1 shows nocarbonyl absorption other than that of the carboxylate at 1600 cm⁻¹,unlike the FTIR of the product in U.S. Pat. No. 5,204,457 which wasprepared with chloroacetic acid.

Example 6

[0092] Carboxymethyl Reduced Dextran CMRD T10 Prepared by the HighDextran Concentration Method.

[0093] Sodium borohydride (0.4 g) and 0.5 g of a 50% solutionweight/weight of sodium hydroxide in water were added to a solution of25 g dextran in 50 g water. The mixture was stirred 4 hours at roomtemperature, 19.5 g of the 1:1 sodium hydroxide solution and 6.2 gbromoacetic acid were added, and the temperature was kept below 25° C.using an ice bath. The mixture was then stirred 16 hours at roomtemperature.

[0094] To purify the product, the pH of the mixture was adjusted to pH6.2 using 6 M HCl, and 120 mL ethanol was added. A precipitate formedand was allowed to settle, and the supernatant was removed by decanting.The residue was dissolved in 60 mL water, and 200 mg sodium chloride wasadded, followed by 30 mL ethanol, and the carboxymethyl reduced dextranwas allowed to settle out. The sequence of addition of water and sodiumchloride followed by dissolution of the precipitate and ethanolprecipitation, was repeated an additional two times. The residue wasdissolved in 60 mL water, and 1 liter of ethanol was added. Thecarboxymethyl reduced dextran was again allowed to settle out, and thesolid was collected on a medium frit glass filter. The white solid wasdried 24 hours at 50° C. The yield was 27 g of product having 1108micromoles carboxyl per gram as measured by titration (Table 2).

Example 7

[0095] Carboxymethyl Reduced Dextran T10 Prepared by the High DextranConcentration Method.

[0096] Sodium borohydride (0.4 g) and 0.5 g of 50% sodium hydroxide wereadded to a solution of 25 g dextran in 50 g water. The mixture wasstirred 4 hours at room temperature, 20.0 g 50% of sodium hydroxide and6.95 g of bromoacetic acid were added and temperature was kept below 25°C. using an ice bath while the mixture was stirred for 16 hours at roomtemperature. The product was purified as described in Example 6. Theyield was 23.9 g of product having 1262 micromoles carboxyl per gram asmeasured by titration (Table 2).

Example 8

[0097] Carboxymethyl Reduced Dextran T10 Prepared by the High DextranConcentration Method.

[0098] Sodium borohydride (0.4 g) and 0.5 g of 50% sodium hydroxide wereadded to a solution of 25 g dextran in 50 g water. The mixture wasstirred for 4 hours at room temperature, and 20.67 g of 50% sodiumhydroxide and 7.65 g bromoacetic acid were added while the temperaturewas kept below 25° C. using an ice bath. The mixture was stirred for 16hours at room temperature. The product was purified as described inExample 6. The yield was 24.5 g of product having 1404 micromolescarboxyl per gram as measured by titration (Table 2).

Example 9

[0099] Carboxymethyl Reduced Dextran CMRD T10 Prepared by the HighDextran Concentration Method.

[0100] Sodium borohydride (0.4 g) and 0.5 g of 50% solution of sodiumhydroxide were added to a solution of 25 g dextran in 50 g water. Themixture was stirred for 4 hours at room temperature, and 20.67 g of 50%sodium hydroxide and 7.65 g of bromoacetic acid were added while thetemperature was kept below 25° C. using an ice bath. The mixture wasstirred for 16 hours at room temperature, and the product was purifiedas described in Example 6. The yield was 23.4 g of product having 1528micromoles carboxyl per gram of product as measured by titration (Table2).

[0101] The relationship between amount of bromoacetic acid used in thesynthesis and the resulting incorporation of micromoles of carboxylgroups into dextran was examined using the high dextran concentrationmethod. The relationship was found to be linear (see Table 2 and FIG.3). Reactant masses and carboxymethyl yields for Examples 6 through 9are shown in Table 2. TABLE 2 Conditions for CMRD synthesis extent anddegree of carboxymethylation of the product. bromoacetic dextran acid,micromoles COOH Example mg/g NaOH, mg/g mg/g per g product 6 246 96.061.0 1108 7 243 97.2 67.6 1262 8 240 99.2 73.4 1404 9 238 100.3 77.21528

[0102] Synthesis of Carboxymethyl Reduced Dextran Preparations UsingReduced Dextran T-10 by the Low Dextran High Base Method.

[0103] Examples 10-14 describe the synthesis of carboxymethyl reduceddextrans with varying degrees of substitution starting with a lowconcentration of reduced dextran. In this method, the startingconcentration of reduced dextran was 70 mg/g and the NaOH was at leastabout 107 mg/g. Table 3 shows that the extent of carboxymethylsubstitution increased as the amount of bromoacetic acid used in thereaction increased.

Example 10

[0104] Carboxymethyl Reduced Dextran CMRD T10 Using the Low Dextran HighBase Method.

[0105] Reduced dextran T10 (15 g) was dissolved in 72 mL water, and 72mL of 8M sodium hydroxide was added. The mixture was brought to 25° C.,and a solution of 1.15 g bromoacetic acid in 3 mL of water was added.The mixture was stirred at room temperature for 1 hour, and then addedto a 75 mL volume of crushed ice. The pH of the solution was brought topH 6.0 using 6M HCl. After repeated ultrafiltration against a 3 kDaultrafiltration membrane, the product was lyophilized. The yield was13.25 g of product. The recovered solid, carboxymethyl reduced dextranT10 (sodium salt), showed a carboxyl content of approximately 110micromoles carboxyl per gram as determined by titration (Table 3).

Example 11

[0106] Carboxymethyl Reduced Dextran T10 Using the Low Dextran High BaseMethod.

[0107] Reduced dextran T10 (150 g) was dissolved in 720 mL water, and720 mL of 8M sodium hydroxide was added. The mixture was brought to 25°C., and a solution of 11.5 g bromoacetic acid in 140 mL water was added.The mixture was stirred at room temperature for 1 hour, added to a 750mL volume of crushed ice, and the pH of the solution was brought to pH6.0 with 6M HCl. After repeated ultrafiltration against a 3 kDa MWCOultrafiltration membrane, the product was lyophilized. The yield was126.21 g of recovered solid carboxymethyl reduced dextran T10 (sodiumsalt), having a carboxyl content of approximately 130 micromolescarboxyl per gram product as determined by titration (Table 3).

Example 12

[0108] Carboxymethyl Reduced Dextran CMRD T10 Using the Low Dextran HighBase Method.

[0109] Reduced dextran T10 (150 g) was dissolved in 720 mL water, and720 mL of 8M sodium hydroxide was added. The mixture was brought to 25°C., a solution of 26.6 g bromoacetic acid in 140 mL water was added, andthe mixture was stirred at room temperature for 1 hour and added to a750 mL volume of crushed ice. The pH of the solution was brought to pH6.0 with 6M HCl. After repeated ultrafiltration against a 3 kDa MWCOultrafiltration membrane, the product was lyophilized. The yield was notdetermined. The recovered solid, carboxymethyl reduced dextran T10(sodium salt), showed a carboxyl content of approximately 280 micromolescarboxyl per gram product as determined by titration (Table 3).

Example 13

[0110] Carboxymethyl Reduced Dextran CMRD T10 Using the Low Dextran HighBase Method.

[0111] Reduced dextran T10 (15 g) was dissolved in 72 mL of water, and72 mL of 8M sodium hydroxide was added. The mixture was brought to 25°C., and a solution of 3.45 g of bromoacetic acid in 8 mL water wasadded. The mixture was stirred at room temperature for 1 hour, and thenadded to a 75 mL volume of crushed ice. The pH of the solution wasbrought to pH 6.0 with 6M HCl . After repeated ultrafiltrations against3 kDa MWCO ultrafiltration membranes, the product was lyophilized. Theyield was 9.4 g of recovered solid carboxymethyl reduced dextran T10(sodium salt), having a carboxyl content of approximately 450 micromolescarboxyl per gram product as determined by titration (Table 3).

Example 14

[0112] Carboxymethyl Reduced Dextran CMRD T10 Using the Low Dextran HighBase Method.

[0113] Reduced dextran T10 (150 g) was dissolved in 720 mL of water, and720 mL of 8M sodium hydroxide was added. The mixture was brought to 25°C., and a solution of 58.8 g of bromoacetic acid in 140 mL water wasadded. The mixture was stirred at room temperature for 1 hour, and wasthen added to a 750 mL volume of crushed ice. The pH of the solution wasbrought to pH 6.0 using 6M HCl. After repeated ultrafiltrations againsta 3 kDa MWCO ultrafiltration membrane, the product was lyophilized. Theyield was 127.88 g of the recovered solid carboxymethyl reduced dextranT10 (sodium salt), having a carboxyl content of approximately 580micromoles carboxyl per gram product as determined by titration (Table3).

[0114] Table 3 shows that the extent of carboxymethyl substitutionobserved was a function of the amount of bromoacetic acid used in thereaction. The data show that generally increasing the amount ofbromoacetic acid in the reaction resulted in increasing levels of COOHin the product. The yield of carboxymethyl incorporation was alsoaffected by conditions such as scale of the reaction, for example, as inExamples 13 and 14. TABLE 3 Preparation of CMRDs with varying extents ofcarboxymethylation. micromoles bromoacetic acid COOH/g Example dextranmg/g NaOH mg/g mg/g product 10 75 115.7 5.77 110 11 75 115.7 5.77 130 1273 111.6 16.7 280 13 70 107.2 27.3 450 14 70 107.2 27.3 580

Example 15

[0115] Carboxymethyl Reduced Dextran T10 from a Commercial Source.

[0116] Carboxymethyl reduced dextran was purchased fromAmersham-Pharmacia. The solid showed a carboxyl content of approximately1887 micromoles carboxyl per gram product as determined by titration.

[0117] Examples 16-18 describe synthesis of carboxymethyl dextran fromnative, non-reduced dextran T-10, T-40, and T-70, respectively.

Example 16

[0118] Carboxymethyl Dextran T10.

[0119] The following solutions were prepared and cooled to 5° C.:Solution A: 105.2 g sodium hydroxide in 250 mL water; Solution B: 58.0 gbromoacetic acid in 142.5 mL water; and Solution C: 75.7 g dextran T10in 187.5 mL water.

[0120] To Solution C and Solution A were added sodium hydroxide (14.4 g)dissolved in 187.5 mL water while maintaining the temperature of thesolution below 25° C. Solution B. was added, keeping the temperaturebelow 25° C., and the resulting solution was stirred for 2 hours at roomtemperature, then was neutralized to pH 7.5 with 6M HCl (cooled to 5°C.) while maintaining the solution temperature below 35° C. The mixturewas passed through a 0.2 μm pore size filter, and diluted to 2 liters.The product was purified by repeated ultrafiltration against a 3 kDaMWCO ultrafiltration membrane, 0.2 μm filtered again, and lyophilized.The yield was 53.17 g, and the recovered solid carboxymethyl dextran T10(sodium salt) showed a carboxyl content of approximately 1220 micromolescarboxyl per gram product as determined by titration.

Example 17

[0121] Carboxymethyl Dextran T40.

[0122] The following solutions were prepared and cooled to 5° C.:Solution A: 154 g sodium hydroxide in 480 mL water; Solution B: 77 gbromoacetic acid in 260 mL water; and Solution C: 100 g dextran T40 in400 mL water.

[0123] Solution A was added to Solution C all at once. After 5 min,Solution B was added and the combined solution was stirred for 120 minwhile the temperature was maintained between 20° C. and 25° C. Themixture was neutralized with 6 M HCl, was 0.2 μm filtered, and dilutedto 2 liters. The product was purified by repeated ultrafiltrationagainst 3 kDa MWCO ultrafiltration membranes, was 0.2 μm filtered andwas lyophilized. The yield was 105.1 g of recovered solid carboxymethyldextran T40 (sodium salt), which showed a carboxyl content of about 1390micromoles carboxyl per gram product as determined by titration.

Example 18

[0124] Carboxymethyl Dextran T70.

[0125] The following solutions were prepared and cooled to 5° C.:Solution A: 154 g sodium hydroxide in 480 mL water; Solution B: 77 gbromoacetic acid 260 mL water; and Solution C: 100 g dextran T70 in 400mL water.

[0126] Solution A was added to Solution C all at once. After 5 min,Solution B was added, and the combined solution was stirred, maintainingthe temperature between 20° C. and 25° C. using an ice bath. After 120min, the solution was neutralized with 6 M HCl. The solution was 0.2 μmfiltered, and diluted to 2 liters. The product was purified by repeatedultrafiltration against 3 kDa MWCO ultrafiltration membranes, was 0.2 μmfiltered again and was lyophilized. The yield was 106.9 g of recoveredsolid carboxymethyl dextran T70 (sodium salt), having a carboxyl contentof about 1380 micromoles carboxyl per gram product as determined bytitration.

[0127] General Procedure for the Preparation of SuperparamagneticColloids for Comparison of the Properties of USPIO Preparations Coatedwith Either of Reduced or Non-reduced Polysaccharides.

[0128] Examples 19-26 were conducted to compare polysaccharide coatediron oxide products obtained from pairs of native and reducedpolysaccharides of identical molecular weights. Identical procedureswere utilized for the preparation of USPIO colloids for each pair ofnative and reduced polysaccharide of identical molecular weight. Inparticular, the same polysaccharide to iron ratio and iron concentrationwas used for each molecular weight pair. The polysaccharide to ironratio and iron concentration utilized for each native and reducedpolysaccharide pair were chosen to yield a 0.2 μm filterable USPIO witha diameter of less than 30 ηm and a magnetic susceptibility of greaterthan 20,000×10⁻⁶ cgs with the reduced polysaccharide.

[0129] The general procedure involved addition of excess ammoniumhydroxide to a solution of iron salts (Fe⁺³/Fe⁺²) and polysaccharide,followed by heating, and performing six cycles of ultrafiltrationagainst water using a 100 kDa MWCO membrane filter. Afterultrafiltration, the USPIO preparations formed with reducedpolysaccharide were filtered through a 0.2 μm filter and stored at 4° C.

[0130] It was observed that for iron oxides prepared with a nativepolysaccharide, only the native dextran T10 coated iron oxide wasfilterable through a 0.2 μm filter. The size and magneticsusceptibility, except for those samples containing particulatematerials, were then measured. Particle sizes were determined bymeasurement of dynamic light scattering in a Microtrac® UPA instrument(Honeywell IAC Microtrac, Fort Washington, Pa.) and are reported as themean volume diameter (MVD). Magnetic susceptibility was determined witha Mathey Johnson magnetic susceptibility balance. Iron concentrationswere determined with a bipyridyl assay (Kumar K., J. Liq. Chromatogr.Relat. Technol., 1997, 20, 3351-3364).

Example 19

[0131] Preparation of Reduced Dextran T1 Coated USPIO.

[0132] Reduced dextran T1 (1.7 g) was dissolved in 20 mL water, and asolution of 3 g of ferric chloride hexahydrate and 1.5 g of ferrouschloride tetrahydrate in 32 g water was added. The mixture was purgedwith nitrogen for 30 min, cooled to 5° C., and 12.7 g of 28% ammoniumhydroxide was added with stirring during a 2 min period. The mixture washeated to 60° C., maintained at this temperature for 40 min, thenincubated at 80° C. for 2 h. The product was subjected to six cycles ofultrafiltration against water using a 100 kDa MWCO membrane filter.After ultrafiltration, the product was filtered through a 0.2 μm filterand stored at 4° C. The product was observed to have the followingproperties: the mean volume diameter (determined by use of a MicrotracParticle Size Analyzer) was 18 nm; the magnetic susceptibility was13,323×10⁻⁶ cgs/g Fe.

Example 20

[0133] Preparation of Native Dextran T1 Coated Iron Oxide.

[0134] Native dextran T1 iron oxide was prepared by the method describedabove for the reduced dextran in Example 19 except that native dextranT1 was used instead of reduced dextran T1. The product was observed tohave the following properties: the mean volume diameter (determined byuse of a Microtrac Particle Size Analyzer) was 2764 nm; the magneticsusceptibility was 1,953×10⁻⁶ cgs/g Fe.

Example 21

[0135] Preparation of Reduced Dextran T5 Coated USPIO.

[0136] Reduced dextran T5 (0.45 g) was dissolved in 13 mL water, and asolution of 0.5 g of ferric chloride hexahydrate and 0.25 g of ferrouschloride tetrahydrate in 4.5 g water was added. The mixture was purgedwith nitrogen for 30 min, cooled to 5° C., and 1.42 g of 28% ammoniumhydroxide was added with stirring during a 2 min period. The mixture washeated at 80° C. for 2 h, and was purified as described in Example 19.The product was observed to have the following properties: the meanvolume diameter (determined by use of a Microtrac Particle SizeAnalyzer) was 16 nm; the magnetic susceptibility was 33,943×10⁻⁶ cgs/gFe.

Example 22

[0137] Preparation of Native Dextran T5 Coated Iron Oxide.

[0138] Native dextran T5 iron oxide was prepared by the method describedabove for the reduced dextran in Example 21 except that native dextranT5 was used instead of reduced dextran T5. The product was observed tohave the following properties: the mean volume diameter (determined byuse of a Microtrac Particle Size Analyzer) was 1,916 nm.

Example 23

[0139] Preparation of Reduced Dextran T10 Coated USPIO.

[0140] Reduced dextran T10 (2.7 g) was dissolved in 70 mL water, and asolution of 2.0 g ferric chloride hexahydrate and 1.0 g ferrous chloridetetrahydrate in 27 g water was added. The mixture was purged withnitrogen for 30 min, cooled to 5° C., and 8.5 g of 28% ammoniumhydroxide was added with stirring during a 2 min period. The mixture washeated at 80° C. for 2 h and purified as described in Example 19. Theproduct was observed to have the following properties: the mean volumediameter (determined by use of a Microtrac Particle Size Analyzer) was12 nm; the magnetic susceptibility was 31,743×10⁻⁶ cgs/g Fe.

Example 24

[0141] Preparation of Native Dextran T10 Coated Iron Oxide.

[0142] Native dextran T10 iron oxide was prepared by the methoddescribed above for the reduced dextran in Example 23 except that nativedextran T10 was used instead of reduced dextran T10. The product wasobserved to have the following properties: the mean volume diameter(determined by use of a Microtrac Particle Size Analyzer) was 757 ηm;the magnetic susceptibility was 31,252×10⁻⁶ cgs/g Fe.

Example 25

[0143] Preparation of Reduced Pullulan Coated USPIO.

[0144] Reduced pullulan (0.045 g) was dissolved in 0.4 mL water, and asolution of 0.106 g ferric chloride hexahydrate and 0.05 g ferrouschloride tetrahydrate in 1.3 g water was added. The mixture was purgedwith nitrogen for 30 min, cooled to 5° C., and 0.044 g of 28% ammoniumhydroxide was added with stirring during a 2 min period. The mixture washeated at 80° C. for 0.67 h and purified as described in Example 19. Theproduct was observed to have the following properties: the mean volumediameter (determined by use of a Microtrac Particle Size Analyzer) was20 nm; the magnetic susceptibility was 27,066×10⁻⁶ cgs/g Fe.

Example 26

[0145] Preparation of Native Pullulan Coated Iron Oxide.

[0146] Native pullulan iron oxide was prepared by the method describedabove for the reduced pullulan in Example 25 except that native pullulanwas used instead of reduced pullulan. The product was observed to havethe following properties: the mean volume diameter (determined by use ofa Microtrac Particle Size Analyzer) was 1,184 ηm.

[0147] Properties of Iron Oxide Preparations Obtained Using Reduced inComparison to Native Polysaccharides (comparison of data obtained fromExamples 19-26).

[0148] In general for MRI contrast agents, an iron oxide contrast agentparticle of small size is preferred, for example, a particle having adiameter in the range of 10 to 50 ηn. Further, an iron oxide of greatermagnetic susceptibility, and of greater homogeneity is preferred.

[0149] It is observed from the data of Examples 19-26 that the presenceof a reduced terminal sugar of a polysaccharide (reduced polysaccharide)used to coat an iron oxide had an unexpected and substantial effect onthe diameter of particles of each of the resulting colloids, compared tosimilarly produced iron oxides made using native non-reducedpolysaccharide. Table 4 shows the size of particles formed for each pairof native and reduced polysaccharides, as indicated by the mean volumediameters (MVD). The concentrations of reduced and nativepolysaccharides were kept constant within each molecular weight group.Concentrations were selected to optimize the synthesis of USPIO withreduced polysaccharide. For all polysaccharides, use of the nativenon-reduced polysaccharide consistently produced a larger particle thandid use of the reduced dextran, so that the reduced polysaccharideconsistently gave the preferred smaller particle.

[0150] Further, for each pair of polysaccharides of a given molecularweight that was synthesized and tested, the USPIO preparation coatedwith reduced polysaccharides demonstrated a higher magneticsusceptibility value than the corresponding iron oxide preparationsynthesized with native polysaccharide, except for colloids obtainedwith dextran T10 for which magnetic susceptibilities of reduced andnative coatings were equivalent.

[0151] These data indicate that use of a reduced polysaccharide inpreparation of coated USPIO colloids yields preferred particles of smallsize, without loss of magnetic susceptibility. The data demonstrate thesurprising effect that reduction of the aldehyde of a polysaccharide hasupon the synthesis of a polysaccharide-coated USPIO. TABLE 4 Comparisonof properties of iron oxides made with native or reduced polysaccharidesunder conditions that form a USPIO with reduced ratio of poly- MVD poly-saccharide nm MS^(a) Example saccharide per Fe, g/g reduced nativereduced native 19, 20 dextran T1 1.6 18 2,764 13,323  1,953 21, 22dextran T5 2.9 16 1,916 33,943 ^(b) 23, 24 dextran 4.6 21 757 31,74331,252 T10 25, 26 pullulan 3.9 20 1,184 27,066 ^(b)

[0152] Properties of Iron Oxides Prepared with Native Non-reduced T1, T5and T10 Dextrans of Mean Volume Diameter Less than 30 ηm.

[0153] Examples 27 through 29 show the preparation of iron oxidesobtained from native dextran T1, T5, and T10. Colloids were preparedusing non-reduced (native) dextrans as described for reduced dextrans(Examples 19, 21, and 23), except that the preparation of these nativenon-reduced dextran particles required about 10- to 34-fold more dextranthan their corresponding reduced dextran counterpart to produce ironoxides of corresponding size. The requirement for increased dextranusage is shown by comparing the dextran to iron ratio of the productsfor corresponding molecular weight pairs of iron oxides shown (Table 5).

[0154] The data show that the magnetic properties, and the efficiency ofdextran use during synthesis, of iron oxide particles prepared with eachof native dextrans T1, T5, and T10 were inferior compared withcorresponding properties of particles prepared with each counterpartreduced dextran.

Example 27

[0155] Preparation of Iron Oxide Coated with Native T1 Dextran.

[0156] A mixture of 0.42 g ferric chloride hexahydrate, 0.21 g ferrouschloride tetrahydrate, and 7.27 g water was filtered through a 0.2 μmfilter. A 1.0 g portion of this mixture was added to 10 mL of an aqueoussolution of 0.1 g dextran T1/g water. The mixture was purged withnitrogen before adding 0.22 mL of 28% ammonium hydroxide solution. Themixture was heated at 80° C. for 1 hour, cooled to room temperature andfiltered through a 0.2 μm filter. The product was observed to have thefollowing properties: the mean volume diameter (determined by use of aMicrotrac Particle Size Analyzer) was 27 ηm; the magnetic susceptibilitywas 2325×10⁻⁶ cgs/g Fe.

Example 28

[0157] Preparation of Iron Oxide Coated with Native T5 Dextran.

[0158] Dextran T5 (0.8 g) was dissolved in 9 mL water, and added to 0.63mL of a 0.2 μm filtered solution of 51.8 mg ferric chloride hexahydrateand 25.9 mg ferrous chloride tetrahydrate in 9.2 mL water. The mixturewas purged with nitrogen before adding 1.4 mL 28% ammonium hydroxidesolution. The mixture was heated at 80° C. for 1 hour, cooled to roomtemperature, and filtered through a 0.2 μm filter. The product wasobserved to have the following properties: the mean volume diameter(determined by use of a Microtrac Particle Size Analyzer) was 20 μm; themagnetic susceptibility was 1285×10⁻⁶ cgs/g Fe.

Example 29

[0159] Preparation of Iron Oxide Coated with Native T10 Dextran.

[0160] Dextran T10 (9420 g) was dissolved in 14915 g water. A 14915 gportion of this mixture was filtered through a 0.2 μm filter, and addedto the reaction vessel. Ferric chloride hexahydrate (891 g) wasdissolved in 713 g water. A 1129 g portion was 0.2 μm filtered and addedto the reaction vessel containing the dextran. The mixture was cooled to5° C. with stirring overnight while bubbling nitrogen through themixture. Before the last 30 min. of the nitrogen purge, a 580 g portionof a 0.2 μm filtered solution of 359 g ferrous chloride tetrahydrate in477 g water was added. To this mixture was added 786 g of 28% ammoniumhydroxide solution, cooled to 5° C. The mixture was heated to 80° C.,incubated at 80° C. for 2 hours, and then poured into 80 liters of waterheated to 80° C. The mixture was allowed to cool overnight, 0.2 μmfiltered, and purified by ultrafiltration using a 100 kDaultrafiltration memberane. The product was 0.2 μm filtered. The productwas observed to have the following properties: the mean volume diameter(determined by use of a Microtrac Particle Size Analyzer) was 21 nm; themagnetic susceptibility was 32,712×10⁻⁶ cgs/g Fe. TABLE 5 Magneticsusceptibility and particle size properties of polysaccharide coatediron oxides: a comparison of native dextrans (Examples 27-29) withrespective reduced dextrans (Examples 19, 21, and 23) under conditionsto give particles of less than 30 ηm MVD with maximum magneticsusceptibility Example dextran type dextran/Fe (g/g)^(b) MVD (nm) MS^(a)iron oxides prepared with native dextran 27 dextran T1 55 27 2,325 28dextran T5 44 20 1,285 29 dextran T10 44 21 32,712 iron oxides preparedwith reduced dextran 19 dextran T1 1.6 18 13,323 21 dextran T5 2.9 1633,943 23 dextran T10 4.6 12 31,743

[0161] Preparation USPIOs Coated with Carboxymethyl Native Dextran T10and Carboxymethyl Reduced Dextran T10 Containing Varying Degrees ofCarboxymethylation.

[0162] Examples 30 and 31 describe preparation of USPIO coated withcarboxymethyl native and reduced dextran T10, respectively. Examples32-36 describe the synthesis of USPIO compositions coated withcarboxymethyl reduced dextran T10 preparations, containing varyingdegrees of carboxymethylation. Examples 37-41 describe the solubility ofpreparations containing ferric chloride and carboxymethyl reduceddextran T10 containing varying degrees of carboxymethylation.

Example 30

[0163] Preparation of USPIO Coated with Carboxymethyl Dextran T10.

[0164] Carboxymethyl dextran T10 (60 g, prepared by the method Example16) was dissolved in 532 g water. A solution of 14.7 g ferric chloridehexahydrate, 7.2 g ferrous chloride tetrahydrate, and 100 mL water, wasfiltered through a 0.2 μm, and added. The mixture was cooled to 10° C.,purged with nitrogen, and 52.2 mL of 28% ammonium hydroxide solution wasadded with stirring. The mixture was heated to 75° C., maintained at 75°C. for 30 min, diluted with 2.5 liter water, and filtered through a 0.2μm filter. The product was purified by repeated ultrafiltration againsta 100 kDa MWCO membrane, concentrated to 20 mg Fe/mL, and again filteredthrough a 0.2 μm m filter. The product was observed to have thefollowing properties: MVD (determined by use of a Microtrac ParticleSize Analyzer) was 19 nm; the magnetic susceptibility was 27,835×10⁻⁶cgs/g Fe; and the carboxyl content was 1,220 micromoles per gram of theCMRD. To determine stability in response to autoclaving, a sample of theproduct was placed in a sealed 5 mL glass vial, and heated to 121° C.for 30 min (see Table 9).

Example 31

[0165] Preparation of USPIO Coated with Carboxymethyl Reduced DextranT10.

[0166] Reduced carboxymethyl dextran T10 (40 g prepared in Example 5)was dissolved in 1,038 mL water and was filtered through a 0.2 1 μm poresize filter. A 0.2 μm filtered solution of 30 g ferric chloridehexahydrate and 15 g of ferrous chloride tetrahydrate in 374 mL of waterwas added to the dextran, with a 31 mL water wash. The solution wascooled to 10° C., and 114 g of 28% ammonium hydroxide was added. Thecolloidal mixture was heated to 78° C. and maintained at thattemperature for one hour. The solution was then diluted to 3 liter withwater, cooled to 10° C., and ultrafiltered 6 times with a YM-100 filtermembrane (100 kDa MWCO). A final concentration of 21.1 mg Fe/g wasobtained. The product was observed to have the following properties: themean volume diameter (Microtrac Particle Size Analyzer) was 21 nm; themagnetic susceptibility was 32,732×10⁻⁶ cgs/g Fe; and the carboxylcontent was 1,265 micromoles per gram of the CMRD. The content of theparticle was determined to be about 50% Fe and 50% dextran. To determinestability in response to autoclaving, a sample of the product was placedin a sealed 5 mL glass vial, and heated to 121° C. for 30 min (see Table9).

Example 32

[0167] Preparation of USPIO Coated with Carboxymethyl Reduced DextranT10 Having 110 Micromoles Carboxyl Per Gram.

[0168] Carboxymethyl reduced dextran T10 (4 g, prepared in Example 10)was dissolved in 85 mL water. To this was added a 0.2 μm filteredmixture of 2.99 g ferric chloride hexahydrate, 1.49 g ferrous chloridetetrahydrate, and 37.3 mL water. The mixture was cooled to 10° C.,purged with nitrogen, 11.4 g of 28% ammonium hydroxide solution wasadded with stirring the mixture was heated to 90° C., maintained at 78°C. for 60 minutes, and then maintained at 78° C. while bubbling airthrough the mixture. The mixture was diluted with 1.5 liters of water,and was filtered through a 0.2 μm filter. The product was purified byrepeated ultrafiltration against a 100 kDa MWCO membrane and againfiltered through a 0.2 μm filter.

Example 33

[0169] Preparation of USPIO Coated with Carboxymethyl Reduced DextranT10 having 130 Micromoles Carboxyl Per Gram.

[0170] Carboxymethyl reduced dextran T10 (40 g, prepared in Example 11)was dissolved in 850 mL water. To this was added a 0.2 μm filteredmixture of 29.9 g ferric chloride hexahydrate, 14.9 g ferrous chloridetetrahydrate, and 373 mL water. The mixture was cooled to 10° C., purgedwith nitrogen, 114 mL of 28% ammonium hydroxide solution was added withstirring, the mixture was heated to 90° C., maintained at 78° C. for 60min, and then maintained at 78° C. while bubbling air through themixture. The mixture was diluted with 1.5 liters of water, and filteredthrough a 0.2 μm filter. The product was purified by repeatedultrafiltration against a 100 kDa MWCO membrane, concentrated to 20 mgFe/mL, and again filtered through a 0.2 μm filter.

Example 34

[0171] Preparation of USPIO Coated with Carboxymethyl Reduced DextranT10 having 280 Micromoles Carboxyl Per Gram.

[0172] Carboxymethyl reduced dextran T10 (4 g, prepared in Example 12)was dissolved in 85 mL water. To this was added a 0.2 μm filteredmixture of 2.99 g ferric chloride hexahydrate, 1.49 g ferrous chloridetetrahydrate, and 37.3 mL water. The mixture was cooled to 10° C., andpurged with nitrogen. To the mixture was added with stirring 11.4 g of28% ammonium hydroxide solution, the mixture was heated to 90° C.,maintained at 78° C. for 60 min, and then maintained at 78° C. while airwas bubbled through the mixture. The mixture was diluted with 1.5 litersof water, and filtered through a 0.2 μm filter. The product was purifiedby repeated ultrafiltration against a 100 kDa MWCO membrane, followed byfiltration through a 0.2 μm filter.

Example 35

[0173] Preparation of USPIO Coated with Carboxymethyl Reduced DextranT10 having 450 Micromoles Carboxyl Per Gram.

[0174] Carboxymethyl reduced dextran T10 (4 g, prepared in Example 13)was dissolved in 85 mL water. To this was added a 0.2 μm filteredsolution of 2.99 g ferric chloride hexahydrate, 1.49 g ferrous chloridetetrahydrate, and 37.3 mL water. The mixture was cooled to 10° C., andpurged with nitrogen before adding 11.4 g of 28% ammonium hydroxidesolution with stirring. The mixture was heated to 90° C., maintained at78° C. for 60 min,and then maintained at 78° C. while air was bubbledthrough the mixture. The mixture was diluted with 1.5 liters of water,filtered through a 0.2 μm filter, and was purified by repeatedultrafiltration against a 100 kDa MWCO membrane followed by filtrationthrough a 0.2 μm filter.

Example 36

[0175] Preparation of USPIO Coated with Carboxymethyl Reduced DextranT10 having 580 Micromoles Carboxyl Per Gram.

[0176] Carboxymethyl reduced dextran T10 (40 g, prepared in Example 14)was dissolved in 85 mL water. To this was added a 0.2 μm filteredsolution of 29.9 g ferric chloride hexahydrate, 14.9 g ferrous chloridetetrahydrate, and 373 mL water. The mixture was cooled to 10° C., purgedwith nitrogen, 11.4 g of 28% ammonium hydroxide solution with stirring.The mixture was heated to 90° C., maintained at 78° C. for 60 min, thenmaintained at 78° C. while bubbling air through the mixture. The mixturewas diluted with 1.5 liters of water and filtered through a 0.2 μmfilter, and was purified by repeated ultra-filtration against a 100 kDaMWCO membrane followed by filtration through a 0.2 μm filter.

[0177] The effect of degree of carboxymethylation of the CMRD coatedUSPIOs on colloid size was compared. Examples 31-36, Table 6. The MVDvalues of the resulting colloids were reasonably uniform between CMRDpreparations containing 110 to 1265 micromoles of carboxyl per gram ofproduct. TABLE 6 Particle sizes of USPIO colloids prepared with dextranT10 CMRDs having varying degrees of carboxymethylation. mean volumediameter, Example # micromoles COOH/g dextran nm 32 110 12 33 130 15 34280 18 35 450 16 36 580 20 31 1265 21

Example 37

[0178] Mixing of Carboxymethyl Reduced Dextran T10 having 1,108Micromoles Carboxyl Per Gram with Ferric Chloride Solution.

[0179] As a step in particle synthesis, ferric chloride (0.3 g) wasdissolved in 15 mL water and was filtered through a 0.2 μm pore sizefilter. Carboxymethyl reduced dextran (prepared in Example 6) was added,the mixture was shaken, and was cooled to 10° C. No precipitate wasobserved.

EXAMPLE 38

[0180] Mixing of Carboxymethyl Reduced Dextran T10 having 1,262Micromoles Carboxyl Per Gram with Ferric Chloride Solution.

[0181] Ferric chloride (0.3 g) was dissolved in 15 mL water and wasfiltered through a 0.2 μm pore size filter. Carboxymethyl reduceddextran (prepared in Example 7) was added, the mixture was shaken, andwas cooled to 10° C. No precipitate was observed.

Example 39

[0182] Mixing of Carboxymethyl Reduced Dextran T10 having 1,404Micromoles Carboxyl Per Gram with Ferric Chloride Solution.

[0183] Ferric chloride (0.3 g) was dissolved in 15 mL water and wasfiltered through a 0.2 μm pore size filter. Carboxymethyl reduceddextran (prepared in Example 8) was added, the mixture was shaken, andwas cooled to 10C. No precipitate was observed.

Example 40

[0184] Mixing of Carboxymethyl Reduced Dextran T10 having 1,528Micromoles Carboxyl Per Gram with Ferric Chloride Solution.

[0185] Ferric chloride (0.3 g) was dissolved in 15 mL water and wasfiltered through a 0.2 μm pore size filter. Carboxymethyl reduceddextran (prepared in Example 9) was added, the mixture was shaken, andwas cooled to 5° C. An orange white precipitate was observed.

Example 41

[0186] Mixing of Carboxymethyl Reduced Dextran T10 having 1,887Micromoles Carboxyl Per Gram with Ferric Chloride.

[0187] Ferric chloride hexahydrate (30.3 g) and ferrous chloride (14.8g) were dissolved in 402.9 mL water and filtered through a 0.2 μm poresize filter. Carboxymethyl reduced dextran T10 (40.3 g in 1,033 ml,prepared in Example 15) was added, the mixture was shaken, and wascooled to 5° C. An orange white precipitate was observed.

[0188] The effect of varying the degree of carboxymethylation of CMRDson the first step of the CMRD-USPIO synthesis, i.e., combining theaqueous mixtures of CMRD with the iron chloride solutions, was analyzed.The various CMRD preparations were mixed with iron salts at a fixed ironconcentration, the CMRD preparations differing only in degree ofcarboxymethylation as described in Examples 37-41. From 1,108 to 1,404micromoles carboxyl per gram dextran, the CMRD formed a homogeneousmixture in the presence of ferric chloride (Table 7). TABLE 7Precipitation of CMRDs having varying levels of carboxyl groups afteraddition of iron salts from mixtures of CMRD (25 mg/g solution) andferric chloride (19 mg/g solution). Example # micromoles COOH/g dextranprecipitate 37 1,108 no 38 1,262 no 31 1,265 no 39 1,404 no 40 1,528yes, at 5° C. 41 1,887 yes, at 25° C.

[0189] At greater than 1,404 micromoles carboxyl per gram dextran,addition of ferric chloride under the conditions and concentrations ofthe USPIO synthesis to the CMRD solution produced an orange whiteprecipitate. Even at higher temperatures, where many compounds can besoluble, the precipitates persisted. The data in Table 7 shows thatthere is an upper level in modification of CMRD that can be used in thepreferred method of CMRD-USPIO synthesis.

Example 42

[0190] Synthesis of Iron Oxide Sols and their Stabilization with Nativeand Reduced Dextrans and CMRD: Preparation of a Magnetic Sol.

[0191] To prepare a magnetic sol, 60 g of 28% of ammonium hydroxide at25° C. was added to a solution having 30.0 g ferric chloride hexahydrateand 15.1 g ferrous chloride tetrahydrate in 321 g of water. After 5minutes of mixing, sufficient concentrated HCl was added to obtain a pHof 1.6. The sol was ultrafiltered with a 100 kDa MWCO membrane filter toachieve a pH of 3.25, using water as diluent. The magnetic sol waspassed through a filter of pore size 0.2 μm, then concentrated to 50 mgFe/g, and stored at 5° C. The yield of iron was 55%, and the product wasobserved to have an MVD of 16 nm.

Example 43

[0192] Synthesis of Iron Oxide Sols and their Stabilization with Nativeand Reduced Dextrans and CMRD: Preparation of a Non-magnetic Sol.

[0193] To a solution of 2.9g of ferric chloride hexahydrate in 30 mL ofwater was added 10 mL of 10 M NaOH. The mixture was stirred for 5 min,diluted to 200 mL with water, and the product was collected byfiltration. The residue was again mixed with water and filtered. Theresidue was added to 40 mL water and the pH was adjusted to 2.0. Theproduct was observed to have an MVD of 10 ηm.

Example 44

[0194] Synthesis of Iron Oxide Sols and their Stabilization with Nativeand Reduced Dextrans and CMRD: Coating of a Magnetic Sol with ReducedDextran T10.

[0195] Reduced dextran T10 (60 mg; Example 3) was dissolved in 1.74 mLwater and combined with 0.24 mL of magnetic sol (13 mg Fe) preparedaccording to Example 42. The mixture was incubated for 15 min, and thepH was adjusted to 7.4 with sodium hydroxide. The particle size (MVD)was determined to be 85 ηm.

Example 45

[0196] Synthesis of Iron Oxide Sols and their Stabilization with Nativeand Reduced Dextrans and CMRD: Coating of a Magnetic Sol with NativeDextran T10.

[0197] Native dextran T10 (60.8 mg) was dissolved in 1.74 mL water, andcombined with 0.24 mL of magnetic sol (13 mg Fe) prepared according toExample 42. The mixture was incubated for 15 min and the pH was adjustedto 7.4 with sodium hydroxide. The particle size (MVD) was determined tobe 1,973 ηm.

Example 46

[0198] Synthesis of Iron Oxide Sols and their Stabilization with Nativeand Reduced Dextrans and CMRD: Coating of a Magnetic Sol with CMRD T10.

[0199] 75 mg of CMRD T10 (Example 5) dissolved in 1.34 mL water wasadded to 0.66 mL of magnetic sol (33 mg Fe) prepared according toExample 42. The mixture was incubated for 15 min at 37° C., and the pHwas adjusted to 7.95 (plus or minus 0.4) with sodium hydroxide. Themixture was concentrated using a 300 kDa ultrafiltration filter. Theproduct was observed to have an MVD of 41 ηm.

Example 47

[0200] Synthesis of Iron Oxide Sols and their Stabilization with Nativeand Reduced Dextrans and CMRD: Adjusting the pH of the Magnetic Sol to7.4.

[0201] A magnetic sol as prepared in Example 42 was adjusted to a pH of7.4. A precipitate was observed.

Example 48

[0202] Synthesis of Iron Oxide Sols and their Stabilization with Nativeand Reduced Dextrans and CMRD: Coating of a Non-magnetic Sol with CMRDT10.

[0203] A non-magnetic sol prepared according to Example 43 (35 ml) wasadded drop-wise to 35 mL of a 50 mg/g aqueous solution of CMRD T10prepared according to Example 5. The pH was adjusted to 7.0 with 1 NNaOH, the solution was heated to boiling, cooled to room temperature,and was centrifuged at 6,000 rpm for 20 min. The supernatant was passedthrough a filter having a 0.2 μm pore size, and autoclaved at 121° C.for 30 min. The product was observed to have a MVD of 86 ηm.

[0204] Examples 42-48 show that in the absence of a dextran, or in thepresence of a native dextran, a gross iron precipitate forms. Onlyreduced dextran and CMRD yielded a magnetic sol as a stable colloid.

Example 49

[0205] Preparation of CMRD Coated Non-magnetic Iron Oxide Colloid UsingBase Co-precipitation of ferric Chloride and CMRD.

[0206] Carboxymethyl reduced dextran T10 (19.2 g) (Example 5) wasdissolved in 300 g water, was filtered through a 0.2 μm filter, and anadditional 160.8 g of water was added. This solution was added to 120 mLof 0.2 μm filtered aqueous 0.3 M ferric chloride hexahydrate. To thismixture was added 32 mL of aqueous 6N sodium hydroxide. The mixture washeated to 100° C. for 3 hours, cooled to room temperature, andultrafiltered to a final volume of 50 mL. The product was observed tohave an MVD of 30 ηm. A portion of this material was placed in a bottleunder nitrogen for 30 min at 121° C. The autoclaved product had an MVDof 69 ηm.

Example 50

[0207] Effect of Autoclaving on Reduced and Native Dextran Colloids:Stability to Autoclaving of USPIOs Coated with Native Dextran andReduced Dextran and CMRD.

[0208] Colloid preparations, each at a concentration of 20 mg Fe/g, wereautoclaved for 30 min at 121° C. Following autoclaving, measurementswere made of bound dextran that was calculated as the difference betweentotal and free dextran, using a phenol/sulfuric acid assay. Free dextranwas separated from the colloid by ultrafiltration. Table 8 shows thatcolloid preparations having USPIOs coated with a reduced dextran havegreater stability than USPIOs coated with a native dextran. The reduceddextran coated USPIO maintained its small size following autoclaving, asthe MVD of the post autoclaved material was increased only 1.3-foldcompared to the MVD of the pre autoclaved material. In contrast, USPIOcoated with native dextran increased in size 28-fold followingautoclaving. The data show that following autoclaving, reduced dextranremains more tightly bound to the iron particle compared to nativedextran.

[0209] A second type of increased stability achieved herein by use ofreduced dextran to coat USPIO is the property of pH of the bulk solvent.The pH of USPIO coated with reduced dextran dropped 0.9 pH unitsfollowing autoclaving, compared to a drop of 1.6 pH units for USPIOcoated with native dextran.

[0210] Even greater stability to the autoclaving process was observedfor particles coated with carboxymethyl reduced dextran compared tocarboxymethyl native dextran. The data in Table 9 indicate that USPIOcoated with carboxymethyl non-reduced native dextran showed a 10-50 foldincrease in amount of particulate matter following autoclaving. Incontrast, USPIO coated with carboxymethyl reduced dextran experienced nochange in size or quantity of particulate matter upon autoclaving.Another indication of the stabilizing effect that the carboxymethylreduced polysaccharide coatings confer on the colloid suspension andbulk solvent was the stability of the solvent pH. The data in bothTables 8 and 9 show that the particles coated with reduced dextran hadsignificantly improved pH stability upon autoclaving, compared to thosecoated with native dextran. TABLE 8 Effect of autoclaving on pH, size,and bound polysaccharide of colloids coated with native and reduceddextran. pre autoclaved post autoclaved^(a) bound bound dextran dextranMVD dextran MVD Example coating pH g/g nm pH g/g nm 29 native T10 7.00.79 21 5.5 0.56 587 23 reduced T10 7.4 1.26 18 6.7 0.96 23

[0211] TABLE 9 Effect of autoclaving on pH, size, and particulates ofcolloids coated with carboxymethylated reduced and carboxymethylatednative non-reduced dextran. particulates^(a) >10 >25 microns micronsExample dextran MVD pH number/ml number/ml autoclaved coating pre postpre post pre post pre post 30 CMD^(b) 19 18 7.5 6.8 35 433 5 240 31CMRD^(c) 25 18 8.0 7.9 4 7 1 5

Example 51

[0212] Procedures for Determining Relaxation Properties of VariousContrast Agents.

[0213] Nuclear magnetic (NM) measurements (0.47T) were obtained in aBruker Instruments pc120 table-top NM sample analyzer operating at 20MHZ (Proton). Half a milliliter of each sample was placed in the 10 mmNM tubes for relaxivity measurements on the minispec. The placement ofthe sample in the sample chamber was optimized. The standards were runand their values recorded in the log.

[0214] Standard procedures were used for T1 and T2 determinations, andtheir values were recorded. T1 was measured using an inversion recoverytechnique. According to the IR technique, the sample is exposed to a180° pulse and then a 90° pulse to put the magnetization in the plane ofdetection. After sampling, the time between the 180 and 90-degree pulsesis changed, and sampled again. This is done for several durations. Theresulting signals are governed by the equation [M_(∞)−M(t)]/M_(∞)=(1-cosθ) exp(−t/T1). When a 3 parameter fit to data is performed, M_(∞),θ, andT1 are calculated.

[0215] T2 was measured using the CPMG technique, where a linear train of180° pulses of variable length is provided to the sample. The amplitudeof every second echo is measured. A fit is performed on the accumulateddata using a two parameter (M_(o) and T2) fit. WhereM(t)=M_(o)exp(−t/T2), a plot of ln(M(t)) versus t is linear with a slopeof −1/T2. The inverse of the T1 and T2 was graphed with respect to theiron concentration of the sample. From the slope of best fit line therelaxivity was determined. TABLE 10 Relaxivity Suscepti- MW R2/ MaterialCoating bility (kDa) R1 R2 R1 Example 31 reduced 38,200 10 35.3 64.8 1.8carboxymethyl dextran Combidex ® Dextran-T10 28,000 9.6 21.7 60.3 2.8Gd-DTPA 172 4.5 5.7 1.3

Example 52

[0216] Toxicity Studies in Rats. Toxicity of Reduced Dextran,Non-reduced Dextran, and CMRD Coated Colloids in Rats.

[0217] An anaphylactic shock type of reaction to dextran can beexhibited by rats and by a small but significant fraction of the humanpopulation (Squire, J. R. et al., “Dextran, Its Properties and Use inMedicine,” Charles C. Thomas, Springfield, Ill., 1955). The reactionresembles anaphylactic shock but does not require prior sensitization,and is characterized in rats by the rapid development of prostration,diffuse peripheral vasodilation, and edema of paws, snout and tongue(Voorhees, A. B. et al., Proc. Soc. Exp. Biol. Med. 1951,76:254). Whenaccompanied by barbiturate anesthesia, it produces marked hypotensionand cyanosis (Hanna, C. H. et al., Am. J. Physiol. 1957,191:615).

[0218] A procedure to measure the extent of rat paw edema response wasemployed to determine if the presence of reduced dextrans or theirderivatives, rather than non-reduced native dextrans, in the coating ofthe iron oxide colloids could decrease or eliminate potential humanadverse reactions upon intravenous injection. Rat paw edema was measuredas the volume of the paw prior to and subsequent to injection of testmaterial, using a plethysmometer, which is a differential volumemeasuring device. The dose of test material was injected, and a secondreading was taken after a designated interval, and the per cent changein paw volume was calculated. The dose administered in these studies was100 mg Fe/kg body weight, a dose much greater than that used as animaging agent in rats, pigs, and humans (see Examples 53-56).

[0219] The results observed following administration of iron oxidescoated with each of reduced and non-reduced T10 dextrans are shown inTable 11. A marked decrease in the edematous anaphylactic response wasobserved in those rats which were administered a USPIO preparationhaving the reduced dextran or reduced dextran derivatives as a coating,compared to those rats administered a USPIO preparation having a nativenon-reduced dextran coating. TABLE 11 Effect of native and reducedpolysaccharide coated particles on rat edema. Example coating andparticle % edema 29 native dextran coated USPIO >50 23 reduced dextrancoated USPIO 13 30 carboxymethyl native dextran coated USPIO 39 48carboxymethyl reduced dextran non-magnetic 12 colloid 31 carboxymethylreduced dextran coated USPIO 0

[0220] The effect of the CMRD-USPIO preparations having increasinglevels of carboxmethyl substitution on the extent of anaphylacticresponse, measured as percent edema, is shown in Table 12. The data showthat a threshold level of substitution was necessary to reduce theedematous response, and that once this threshold of substitution wasachieved, the decrease in response of the rats to dextran was asurprising elimination of the edematous response. That is, no edema wasobserved at 1,265 micromoles of carboxyl per gram. TABLE 12 Extent ofrat paw edema as a function of amount of carboxymethylation of dextrancoating of USPIOs. micromol COOH Example per g dextran % edema 32 110 2433 130 54 34 280 81 35 450 37 36 580 105 31 1,265 0

Example 53

[0221] Toxicity Studies in Rats of Reduced and Non-reduced Dextrans.

[0222] The procedure used in Example 52 was used to determine if thecoating alone, that is, reduced dextrans or their derivatives ratherthan non-reduced native dextrans, could eliminate potential humanadverse reactions upon intravenous injection. Rat paw edema was measuredas the volume of the paw prior and subsequent to injection, as inExample 52. The dose administered in these studies was, as above, 100 mgtest substance/kg body weight.

[0223] The results observed following administration of reduced andnon-reduced T10 dextrans were similar for each material (Table 13).Reduced dextran T10 elicited the same extent edema as native dextranT10. Elimination or decrease in edema could not be attributed merely toreduction of the dextran. TABLE 13 Effect of native and reduced 10 kDapolysaccharides on rat edema showing mere reduction has no significanteffect % Example test dextran edema Dextran T-10 native T10 61(commercial^(a)) 3 reduced T10 67

[0224] Table 14 shows the effect of increased levels of carboxymethylsubstitution of reduced dextran on the extent of anaphylactoid response,measured as percent edema. The data show that above a threshold level ofcarboxymethyl substitution, edema was decreased or eliminated. Fordextrans above this threshold level of substitution, the decrease in thetoxic response of the rats to dextran was a surprising elimination ofresponse, that is, no edema was observed. TABLE 14 Relationship betweenrat paw edema and degree of carboxymethylation of dextran T10preparations. micromol Example test substance COOH/g per dextran % edema10 carboxymethyl reduced 110 65 12 carboxymethyl reduced 280 60 13carboxymethyl reduced 450 56  5 carboxymethyl reduced 1,265 6 15carboxymethyl reduced 1,887 1 16 carboxymethyl native 1,220 0

Example 54

[0225] Pharmacokinetics of CMRD Coated USPIO in the Rat: BloodClearance.

[0226] Three male CD® rats (Charles River Laboratories, Wilmington,Mass.; weight range 272 to 290 g) were anaesthetized intraperitoneallywith a long lasting anesthetic, Inactin (100 mg per body weight). Thefemoral artery and vein were exposed by a small incision at thehip-femur joint, and the artery was cannulated with PE50 tubingconnected to a 1 mL syringe filled with heparinized saline (10 units perml). To serve as a baseline, 0.25 mL of arterial blood was collected attime zero, and CMRD coated USPIO (Example 31) was injected into thefemoral artery. Blood samples of 0.25 mL were collected at the timesindicated in FIGS. 4 and 5 .

[0227] T2 magnetic relaxation times were measured in each sample, andthe relaxivity (1/T2) was calculated. First-order reaction kinetics wereused to determine the half-life of the sample in the blood (t_(1/2)).The equation used to fit the data was:

1/T2−1/T _(baseline) =A e ^(−kt)

[0228] where 1/T₂ is the relaxivity of the blood at time tpost-injection; 1/T_(baseline) is the baseline relaxivity, and A e^(−kt)represents the first-order decay of the test material from the blood.Taking the natural log of each side of this equation yields:

ln (1/T2-1/T_(baseline))=−kt+lnA ₀

[0229] According to this second equation, a graph of ln(1/T2−1/T_(baseline)) versus time, t, should give a straight line withslope −k (the first order rate constant) and intercept ln A₀ (whichequals ln (1/T2−1/T_(baseline) at time zero) if the rate of removal ofthe USPIO from blood follows first order kinetics. FIG. 5 shows that astraight line was obtained. The half-life (t_(1/2)), which is the timethat the amount of CMRD coated USPIO decreased to one half its amount ofconcentration in the blood, was determined to be 67 min, with a range of61 to 75 min at a confidence level of 95%.

Example 55

[0230] Magnetic Resonance Imaging Using CMRD Coated USPIO in the Rat.

[0231] An MRI scan of a rat taken shortly after administration of 5 mgof CMRD coated USPIO (Example 31) per kg body weight is shown in FIG.6B. The heart, aorta, and coronary artery were found to be readilyimaged using this agent. An image of the rat taken pre-administration ofthe agent (FIG. 6A) is included to illustrate the substantial increasein contrast effected by administration of the test substance.

Example 56

[0232] MRI of CMRD Coated USPIO in the Pig.

[0233]FIG. 7 illustrates enhanced MRI visualization of the heart andsurrounding arteries, as well as the lungs and kidneys of the pig. Fourdoses of 0.4, 0.8, 1.6, and 2.2 mg of iron/kg body weight of sample(Example 31) were each administered to the pig in sequential order. Eachdose was followed by administration of 20 mL of physiological saline,and an MRI image was obtained after each dose. The image shown in FIG.7B is representative of images obtained after each administration. Apreimage of the pig (FIG. 7A) is included to illustrate the substantialincrease in contrast effected by the agent.

[0234] A problem associated with low molecular weight gadolinium basedcontrast agents is that they leak from the vascular space into theinterstitial space and create a hazy background. This hazy backgroundinterferes with effective use of second or third injections of acontrast agent administered during a single examination. Suchextravascular leakage might not be expected with carboxymethyl reduceddextran-coated USPIOs due to the relatively large size of the particle,compared to the size of the particles of a gadolinium contrast agent.

[0235] This expectation was confirmed by imaging of rats (Example 55)and in the data obtained by imaging of the pig (FIG. 7B). No backgroundhaze was observed following use of the CMRD USPIO compositions of thepresent invention. This observation enabled performance of additionalvascular imaging tests, after sequential administration of additionaldoses. Upon intravenous administration, the CMRD coated USPIO, which isan embodiment of the invention, moved as a bolus rapidly into thearteries, organs, and veins, and achieved a uniform distribution in theblood after 20 minutes. Upon administration of a second bolus of theagent, additional good images were obtained. A third injection and afourth injection were administered with similar results i.e., goodimages were obtained. Thus, the process of bolus injection and firstpass application of the CMRD coated USPIO was demonstrated. Further,application of a multiple injection protocol within a reasonably shortperiod of time after the first administration, the entire protocol beingaccomplished in a time period equivalent to a visit by a human subjectto an imaging facility, was also demonstrated.

[0236] The principal advantages of capability of multiple bolusinjections within a single examination are the opportunities to correcta deficiency in imaging that might arise after an injection, and toimage multiple parts of the body during a single examination. In thismanner, additional sites within the body of a subject can be imagedwithin a short period of time after scanning and analysis of earlierimages from an earlier pass, and subsequent injections of contrast agentcan be used to obtain different views, or to extend the view in one ormore physical dimensions. For example, detailed analysis of the locationand size of a blood clot in a limb such as a leg, can be performed usinga series of views taken in the each of a first, second, and subsequentpasses.

[0237] The capability for achieving additional multiple passes ofadministration of a composition of the invention and obtainingadditional rounds of MRI data, beyond a first dose, present strongadvantages of the compositions that are embodiments of the presentinvention. MRI analyses have in the past been limited by the physicallength of the anatomical feature in need of imaging, and by the numbersof structures that can be imaged using a single detection instrumentunit in a given time period. The results obtained in pigs were observedalso in human subjects (Examples 57 and 58).

Example 57

[0238] Intravenous Injection of CMRD Coated USPIO into Normal HumanSubjects.

[0239] The trial design employed thirty-five human subjects eachadministered one dose of CMRD T10 coated USPIO prepared according toExample 31(i.v.; 1-4 mg of iron/kg body weight). The objectives ofExamples 57 and 58 were to examine subjects for any potential sideeffect of the treatment, to obtain data on the composition as an MRIcontrast agent, and to determine the half-life of the composition inblood.

[0240] No adverse reactions attributable to administration of thecomposition were observed among the treated subjects at any dose,including the highest dose (4 mg/kg). For comparison, in clinical trialsof Feridex I.V.®, approximately 2-3% of treated patients reported backpain, even though Feridex I.V.® and other comparable imaging productsare administered in much smaller doses (e.g., 0.56 mg of iron/kg bodyweight) in order to minimize adverse events and obtain useful contrast.These data indicate that an effective dose of the CMRD coated USPIOparticles of the invention is safer than an effective dose of apreviously approved imaging agent, Feridex I.V.®

Example 58

[0241] Rapid Imaging Kinetics and Bio-distribution in Human Subjects.

[0242] An initial intravenous bolus injection into human subjects ofCMRD coated USPIO, prepared as in Example 31 yielded a bright MRI of thearterial portion of the circulatory system within 12 secondspost-administration (FIG. 8B). Following a further 15 seconds, MRIexposures yielded bright images of organs and veins. Equilibration ofthe agent throughout the vascular system was achieved within 20 minutes.

[0243] The organs capable of being imaged in the early phase followingadministration of the CMRD coated USPIO of the present inventionincluded the heart, arteries and veins. Further, in addition to thelarger elements of the circulatory system, the arterioles and venules ofthe extremities (fingers, toes) could be observed. This level ofresolution allows applications to diagnosis of problems in circulationwithin the extremities, including the detection and localization of anarea of phlebitis. Other organs that were readily imaged include thebrain, kidneys, liver, spleen, and bone marrow. Lymph nodes could beimaged up to several hours after administration of an effective dose.The half-life of the agent in the blood was approximately observed to be10-14 hours (see Table 15 and FIG. 9).

[0244] The particles ultimately were removed from circulation by beingtaken up by the reticuloendothelial system. During the presence of thecomposition at the early phase in the vascular system, and also in thelate or post vascular phase in the reticuloendothelial system (RES),this composition was not observed to enter into interstitial spacesbetween cells. Thus, a hazy background, found to appear with usage ofother compositions, for example, gadolinium based MR contract agentssuch as Magnevist® and DOTOREM®, is avoided during use of the CMRD-USPIOcompositions, as synthesized by the methods of the Examples herein.TABLE 15 Mean half-life of CMRD-USPIO T10 in human subjects as afunction of dose. Dose mg iron/kg half-life, hours standard deviation #subjects 1 9.7 1.1 8 2 10.3 1.4 8 4 14.4 2.2 17

What is claimed is:
 1. An improved method of administering to amammalian subject a polysaccharide iron oxide complex, wherein theimprovement comprises administering a carboxyalkylated reducedpolysaccharide iron oxide complex having an extent of carboxyalkylationsufficient to produce decreased edematous response to thecarboxyalkylated complex in comparison to an edematous response to anadministered polysaccharide that has not been thus carboxyalkylated. 2.An improved method of administering to a mammalian subject apolysaccharide wherein the polysaccharide is dextran and wherein theimprovement comprises administering a carboxyalkylated reduced dextranhaving an extent of carboxyalkylation sufficient to produce decreasededematous response to the carboxyalkylated dextran in comparison to anedematous response to an administered dextran that has not been thuscarboxyalkylated.
 3. A method according to claim 2, further comprisingsterilizing the polysaccharide by autoclaving.
 4. A method according toclaim 3, wherein the subject is in need of a plasma extender.
 5. Amethod according to claim 1, further comprising providing a solution ofan iron salt to form a carboxyalkylated reduced dextran iron colloidformulation producing decreased edematous response.
 6. A methodaccording to claim 1, further comprising sterilizing thecarboxyalkylated reduced dextran iron oxide complex by autoclaving.
 7. Amethod according to claim 6, wherein the subject is in need of iron. 8.A method according to claim 7, wherein the subject in need of iron isselected from the group of: a cancer patient, a gastroenteritis patient,and an erythropoietin recipient.
 9. An improved method of magneticresonance imaging (MRI) wherein the improvement comprises administeringto a subject an effective dose of a carboxyalkylated reducedpolysaccharide iron oxide MRI contrast agent to facilitate magneticresonance imaging (MRI) of a tissue or organ wherein upon administeringthe carboxyalkylated reduced polysaccharide MRI contrast agent there isa decreased edematous response in comparison to an administeredpolysaccharide MRI contrast agent that has not been thuscarboxyalkylated.
 10. An improved method of magnetic resonance imaging(MRI), wherein the improvement comprises administering to a subjectsuccessive effective doses of a carboxyalkylated reduced polysaccharideiron oxide MRI contrast agent to facilitate successive magneticresonance imaging of a tissue or organ wherein upon administering thecarboxyalkylated reduced polysaccharide MRI contrast agent there is adecreased edematous response in comparison to an administeredpolysaccharide MRI contrast agent that has not been thuscarboxyalkylated.
 11. A method according to claim 9, wherein theeffective dose is about 0.1 to about 4.0 mg of iron per kg of bodyweight of the subject.
 12. A method according to claim 11, wherein theeffective dose is about 0.2 to about 0.6 mg of iron per kg of bodyweight.
 13. A method according to claim 11, wherein the effective doseis about 0.4 to about 1.0 mg of iron per kg of body weight.
 14. A methodaccording to claim 11, wherein the effective dose is about 1.0 to about4.0 mg of iron per kg of body weight.
 15. A method according to claim10, wherein the successive effective doses are administered less thanone hour apart.
 16. A method according to claim 15, wherein thesuccessive effective doses are administered less than thirty minutesapart.
 17. An improved method for obtaining a composition forpharmacological administration from a polysaccharide, wherein theimprovement comprises prior to administering: reducing thepolysaccharide; and carboxyalkylating to an extent sufficient to producea decreased edematous response in the subject to the administeredpolysaccharide composition in comparison to an edematous response in thesubject to an administered polysaccharide that has not been thus reducedand carboxyalkylated.
 18. A method according to claim 17, wherein themethod further comprises: administering the reduced carboxyalkylatedpharmacological polysaccharide composition to a mammalian subject as aplasma extender.
 19. An improved method for obtaining a composition forpharmacological administration from a dextran, wherein the improvementcomprises: reducing the dextran; and, carboxyalkylating the dextran toprovide an extent of carboxyalkylation sufficient to produce a decreasededematous response to the administered dextran composition in a subjectin comparison to an edematous response in a subject to an administeredpolysaccharide that has not been thus reduced and carboxyalkylated. 20.A method according to claim 17, further comprising the step ofsterilizing the carboxyalkylated reduced dextran composition byautoclaving.
 21. A method according to claim 20, further comprisingafter sterilizing the step of providing the sterile composition as asingle dosage unit.
 22. A method according to claim 20, furthercomprising the step of administering the composition to a mammal in needof a plasma extender.
 23. A product for use as a plasma extenderproduced by the improved method of claim 20.